Method for influencing pollen development by modifying sucrose metabolism

ABSTRACT

Methods for influencing pollen development by modifying the sucrose metabolism in transgenic plant cells and plants and generating male sterile plants by sucrose depletion in pollen. An expression of a protein has enzymatic activity of a sucrose isomerase in transgenic plant cells. Nucleic acid molecules contain a DNA sequence encoding a protein having the enzymatic activity of a sucrose isomerase, wherein the DNA sequence is functionally linked with the regulatory sequences of a promoter active in plants so that the DNA sequence is expressed in anthers or pollen. The invention further relates to transgenic plants and plant cells that contain the inventive nucleic acid molecule and whose male plant and plant cells are sterile due to the expression of the DNA sequence that encodes a protein having the enzymatic activity of a sucrose isomerase. The invention also relates to harvest products and the propagation material of said transgenic plants.

RELATED APPLICATIONS

[0001] This application is a continuation under 35 U.S.C. 111(a) ofInternational Application No. PCT/EP01/01412 filed Feb. 9, 2001 andpublished in German on Aug. 16, 2001 as WO 01/59135 A1, which claimedpriority from German Application No. 100 45 113.6 filed Sep. 13, 2000and German Application No. 100 06 413.2 filed Feb. 14, 2000, whichapplications and publication are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for influencing thepollen development by modifying the sucrose metabolism in transgenicplant cells and plants. The invention especially relates to a method forgenerating male sterile plants wherein carbohydrates are depleted fromdeveloping pollen. The invention particularly relates to the expressionof a protein having the enzymatic activity of a sucrose isomerase intransgenic plant cells. The present invention further relates to nucleicacid molecules that contain a DNA sequence which codes for a proteinhaving the enzymatic activity of a sucrose isomerase, and wherein theDNA sequence is operatively linked to the regulatory sequences of apromoter active in plants so that the DNA sequence is expressed inanthers or pollen. The present invention also relates to transgenicplants and plant cells which contain the nucleic acid molecule accordingto the invention and, due to the expression of the DNA sequence thatencodes a protein having the enzymatic activity of a sucrose isomerase,are male sterile, as well as harvest products and propagation materialof the transgenic plants.

BACKGROUND OF THE INVENTION

[0003] As eukaryotes, plants possess two or more copies of their geneticinformation per cell. Each gene is generally represented by two alleles,which can be identical in the homozygous state or different in theheterozygous state. When two selected inbreeding lines are crossed, theF1 hybrids formed in the first generation, i.e., heterozygousindividuals, are frequently larger, more robust and therefore moreproductive than the homozygous parents, probably because their twoallelic gene products a) have a lower probability of being inactivatedor b) have a larger reaction width. Plant breeders have used thiseffect, known as heterosis or hybrid vitality, for many decades toproduce hybrid species.

[0004] Such hybrid lines are bred using cytoplasmic male sterility (CMS)or self-incompatibility (SI), the two most important genetic systems forpreventing self-fertilisation.

[0005] The possibility of incorporating a new gene into the genome of aplant cell using gene technological methods has, during the last fewyears, revealed a third possibility of producing hybrid plants, namely,the use of a synthetically male-sterile system.

[0006] Male sterility produced by genetic engineering has already beenachieved by various strategies. These include, among others, theexpression of a ribonuclease (RNase) from Bacillus amyloliquefaciens inthe tapetum (the tapetum is the cell layer which provides the pollencells with nutrients during their development) of tobacco anthers(Mariani C. et al. (1990) Nature 347:737-741; Mariani C. et al. (1992)Nature 357:384-387), the overexpression of the rolC gene fromAgrobacterium rhizogenes in tobacco (Schmülling T. et al. (1988) EMBO J.7:2621-2629; Schmülling T. et al. (1993) Mol. Gen Genet. 237:385-394)and in potatoes (Fladung M. (1990) Plant Breeding 104:295-305) and theexpression of a glucanase, whose activity prematurely destroys thecallose cell wall of the microsporocyte (Worrall D. (1992) Plant Cell4:759-771). Other approaches for the production of male sterile plantsare connected with modifying the pigment composition of the blossombased on isolating and manipulating the genes involved in the flavonoidbiosynthesis. Here the inhibition of a certain step in the flavonoidsynthesis is generally achieved by the anti-sense technique or theexpression of additional sense constructs (see, for example van der KrolA. R. et al. (1988) Nature 333:866-869; Napoli C. (1990) Plant Cell2:279-289; van der Meer I. M. et al. (1992) Plant Cell 4:253-262; TaylorL. B. and Jorgenson R. (1992) J. Heredity 83:11-17). These studies, ofwhich most are concerned with the trans-inactivation of chalconesynthase, confirm the assumption that flavonoids not only contribute tothe blossom or flower colour, but also play an essential role in antherand pollen development.

[0007] In another approach to produce male sterility, an externallyapplied pre-herbicide is converted into a herbicide by the introducedtransgene. Thus, in transgenic tobacco plants which express the argEgene from E. coli under the control of the TA29 promoter, theapplication of the non-toxic substance N-acetyl-L-phosphinotricin duringpollen development results in male sterility (Kriete et al. (1996) PlantJ. 9:809-818). As a result of the activity of the argE gene, thenon-toxic pre-herbicide is deacetylated and converted into the cytotoxicL-phosphinotricin.

[0008] If the hybrid plant produced is a crop plant whose seeds, fruitsor blossoms, i.e. generative organs, are to be harvested, a restorersystem must also be introduced so that the F1 plant is again malefertile. In the case of the afore-mentioned expression of aribonuclease, the ribonuclease activity destroys the function of thetapetum with the consequence that the pollen is no longer viable andmale sterile plants are produced. In this case, a restorer system wasdeveloped based on the expression of a ribonuclease inhibitor gene,which was isolated from the same bacterium (B. amyloliquefaciens) thatexpresses the ribonuclease.

[0009] However, F1 hybrid lines are of particular importance not onlybecause of their increased vitality and yield. The seed-growing andbreeding industry has also acquired major commercial importance becausethe farmer cannot further propagate F1 hybrid species since asegregation of the positive properties occurs in the F2 generation andplants produced from seeds of F1 hybrids have a much lower resistanceand performance than the F1 hybrids. The farmer must therefore buy newseed from the seed producer for each sowing.

[0010] Although intensive research is being carried out on theproduction of gene technologically produced hybrid lines with improvedagronomic properties and less expenditure of work (since mechanicalcastration becomes no longer necessary), in many cases, however, themethods hitherto available for the production of male sterile plants donot yield completely satisfactory results. In addition, plants with aconsiderably increased sensitivity to phytopathogens are frequentlyobtained which makes them extremely difficult to handle in practice.There is thus a strong need for further methods for the production ofmale sterile plants which do not show the disadvantages of the priorart.

SUMMARY OF THE INVENTION

[0011] It is thus an object of the present invention to provideavailable new methods for influencing the pollen development and thusfor the production of male sterile plants, and recombinant DNA moleculeswhich contain a DNA sequence which can be used to manipulate pollendevelopment and, especially here, to produce male sterile plants.

[0012] This and further objects of the invention are achieved byproviding the embodiments characterised in the claims.

[0013] Surprisingly, it has now been found that genes, whose expressionin the anthers leads to a modification of the sucrose metabolism andespecially has the effect that the developing pollen are depleted insucrose and other carbohydrates, are suited to the production of malesterile plants. Especially useful here are DNA sequences which code fora protein having the enzymatic activity of a sucrose isomerase.

[0014] Proteins with sucrose isomerase activity catalyse theisomerisation of the disaccharide sucrose to other disaccharides. Inthis case, the α1→β2-glycosidic bond between the two monosaccharideunits of sucrose, namely the glycosidic bond between glucose andfructose, is converted into another glycosidic bond between twomonosaccharide units. Especially, sucrose isomerases, also known assucrose mutases, catalyse the rearrangement into an α1→6 bond and/or anα1→α1 bond. In this case, the disaccharide palatinose is formed as aresult of isomerisation to an α1→6 bond whereas the disaccharidetrehalulose is formed during the rearrangement to an α1→α1 bond.

[0015] Examples of organisms whose cells contain nucleic acid sequencescoding for a protein having sucrose isomerase activity especiallyinclude micro-organisms of the genus Pro-taminobacter, Erwinia,Serratia, Leuconostoc, Pseudomonas, Agrobacterium, Klebsiella andEnterobacter. Here particular mention may be made of the followingexamples of such micro-organisms: Protaminobacter rubrum (CBS 547, 77),Erwinia rhapontici (NCPPB 1578), Serratia plymuthica (ATCC 15928),Serratia marcescens (NCIB 8285), Leuconostoc mesenteroides NRRL B-521f(ATCC 10830a), Pseudomonas mesoacidophila MX-45 (FERM 11808 or FERM BP3619), Agrobacterium radiobacter MX-232 (FERM 12397 or FERM BP 3620),Klebsiella subspecies and Enterobacter species.

[0016] Now it was surprisingly observed that in transgenic plants, inwhose anthers sucrose is converted into palatinose as a result of theexpression of gene technologically introduced sucrose isomerase DNAsequences, this expression of sucrose isomerase DNA sequences leads to amale sterile phenotype.

[0017] Without wishing to be bound to a hypothesis, the followingexplanation is currently accepted for the phenomenon now observed.Developing pollens are supplied with assimilates (photosynthates) byspecialised cells of the anthers. Carbohydrates are transported into theapoplast in the form of the disaccharide sucrose. For the uptake ofsugars the pollens secrete extracellular invertases, which ensure thatthe hexoses glucose and fructose are produced. These monosaccharides aretaken up by available hexose transporters and are metabolised. As aresult of the expression of a sucrose isomerase in the anthers,palatinose, among others, is formed from sucrose. However, thedisaccharide palatinose can only be cleaved by corresponding hydrolases,but not by the afore-mentioned invertases. This has the result that thepollen cannot develop, with the consequence of male sterility.

[0018] This effect can also be achieved by other measures which resultin a modification of the sucrose metabolism, especially in the depletionof sucrose and utilisable hexoses and thus in an undersupply of thepollen with carbohydrates. Thus, the pollen development can bedisturbed, for example by the inhibition of invertases, hexosetransporters and hexokinases, which leads to the male sterile phenotypeof plants transformed with corresponding nucleic acid molecules. Thedevelopment of functional pollen can also be prevented by the fact thatosmotically active substances are produced in the anthers or accumulatethere, which leads to desiccation of the developing pollen and thus tothe male sterile phenotype.

[0019] In most plants, the carbon-supply of the developing pollen isprovided by sucrose, which was generated in photosynthetically activeleaves and loaded into the conducting tissue (assimilate conductingtissue) of the phloem. The sucrose is secreted by tapetum cells into theapoplast, hydrolysed to glucose and fructose by apoplastic invertasesand are taken up into the pollen by hexose transporters. In the cytosolof the pollen the hexoses are phosphorylated by means of hexokinases andthus made available for metabolism. The hexoses are taken up along withprotons, which are pumped into the apoplast by means of ATPases. Asmentioned above various approaches, which inhibit the uptake andutilisation of monosaccharides are thus possible to interrupt thecarbohydrate supply of the developing pollen.

[0020] The observation that the development of pollen can be effectivelyinhibited by influencing the sucrose metabolism, and here especially bydepleting utilisable monosaccharides, can be ideally used to producemale sterile crop plants. The basic assumption for the production ofsuch male sterile crop plants is the availability of suitabletransformation systems. Over the last two decades a broad spectrum oftransformation methods has been developed and established in this field.These techniques comprise the transformation of plant cells with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes as thetransforming agent, diffusion of protoplasts, the direct gene transferof isolated DNA into protoplasts, injection and electroporation of DNAinto plant cells, introduction of DNA by means of biolistic methods andother possibilities.

[0021] Another prerequisite for the production of plants which expressDNA sequences encoding a protein having the enzymatic activity of asucrose isomerase in their anthers, their tapetum or their pollen andare male sterile as a result of this specific expression, is thatsuitable DNA sequences are available.

[0022] Such sequences from Protaminobacter rubrum, Erwinia rhapontici,Enterobacter species SZ 62 and Pseudomonas mesoacidophila MX-45 aredescribed in PCT/EP 95/00165. Reference is hereby made to the disclosureof this patent application, both with respect to the disclosed sequencesthemselves as well as with reference to the identification andcharacterisation of these and other sucrose isomerase coding sequencesfrom other sources.

[0023] The person skilled in the art can obtain other sucrose isomerasecoding DNA sequences from the relevant literature and gene databasesusing suitable search profiles and computer programs for screening forhomologous sequences or for sequence alignments.

[0024] The person skilled in the art himself can also identify othersucrose isomerase coding DNA sequences from other organisms by means ofconventional molecular biological techniques and use these DNA sequenceswithin the scope of the present invention. Thus, for example, the personskilled in the art can derive suitable hybridisation probes from knownsucrose isomerase sequences and use these probes for screening cDNAand/or genomic libraries of the particular desired organism from which anew sucrose isomerase gene is to be isolated. Here the person skilled inthe art can go back to current hybridisation, cloning and sequencingmethods, which are well-known and established in every biotechnology orgene technology laboratory (see, for example Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The person skilled in theart can, of course, also synthesise and use suitable oligonucleotideprimers for PCR amplifications of sucrose isomerase sequences usingknown sequences.

[0025] The same applies to other measures which result in an undersupplyof developing pollen with carbohydrates and especially in the depletionof utilisable monosaccharides and therefore in male sterile plants. Herealso the various points of attack such as invertases, hexosetransporters and hexokinases are well known to the person skilled in theart from the literature so that he is capable of effectively inhibitingthe corresponding proteins, for example, by transfer of anti-sense orsense or cosuppression constructs and thereby interrupting theutilisation of sucrose in the anthers.

[0026] As targets, here mention can be made of, as noted above: cellwall-bound invertases: here a plurality of genes or cDNA clones can beobtained from the relevant databases and publications, which allow theperson skilled in the art to produce suitable constructs for inhibitingcell wall-bound invertase and to transfer them to plant cells usingroutine methods. Examples of suitable sequences are: Arabidopsis(Schwebel-Dugue et al. (1994) Plant Physiol. 104, 809-810), carrot(Ramloch-Lorenz et al. (1993) Plant J. 4, 545-554), tobacco (Greiner etal. (1995) Plant Physiol. 108, 825-826), tomato (Ohyama et al. (1998)Genes Genet. Syst. 73, 149-157), Vicia faba (Accession No. Z35163),Pisum sativum (Accession No. Z83339), Beta vulgaris (Accession No.X81795). Further sequences can easily be identified by homologycomparisons so that inhibition of the invertases is possible in allrelevant crop plants.

[0027] Besides the inhibition by anti-sense or sense constructs,invertase activity can also be suppressed by expression of invertaseinhibitors. The invertase inhibitor from tobacco is given as example(see Greiner et al. (1998) Plant Physiol. 116, 733-742). Overexpressionof an invertase inhibitor in transgenic plants resulted in inhibition ofendogenous invertase activity in potato tubers (Greiner et al. (1999)Nat. BioTech. 17, 708-711).

[0028] Another target are hexose transporters (monosaccharidetransporters). Here also a plurality of genes or cDNA clones can beobtained from the databases and publications, which allow the personskilled in the art to create constructs for the inhibition ofpollen-expressed hexose transporters. Examples of published sequencesare: petunia (Ylstra et al. (1998) Plant Physiol. 118, 297-304),Arabidopsis (Truernit et al. (1999) Plant J. 17, 191-201), tobacco(Sauer and Stadler (1993) Plant J. 4, 601-610), Medicago sativa(Accession No. AJ248339), Ricinus communis (Accession No. L08191). Othersequences can easily be identified by homology comparisons so thatinhibition of hexose transporters is possible in all relevant cropplants. At this point it should be noted that the carbohydrates that arerequired for the supply of the growing pollen tube during fertilisationmust also be transported (taken up) via a hexose transporter. This meansthat the inhibition of the transporter impedes both the pollen formationand the vitality of the pollen.

[0029] Undersupply of pollen with carbohydrates can also be achieved byinhibition of proton ATPases. Here also a plurality of genes or cDNAclones can be obtained from the databases and publications, which allowthe person skilled in the art to create constructs for the inhibition ofthe plasma membrane-bound proton ATPase. Examples of published sequencesinclude: Vicia faba (Nakajima et al. (1995) Plant Cell Physiol. 36,919-924), potato (Harms et al. (1994) Plant Mol. Biol. 26, 979-988),rice (Ookura et al. (1994) Plant Cell Physiol. 35, 1251-1256). Othersequences can easily be identified by homology comparisons so thatinhibition of the plasma-membrane-bound proton ATPase is possible in allrelevant crop plants.

[0030] Another approach relates to the afore-mentioned hexokinases.Here, the same applies as to the other targets a plurality of genes orcDNA clones can be obtained from the databases and publications whichallow the person skilled in the art to create constructs for theinhibition of hexokinase. Examples of published sequences are: spinach(Wiese et al. (1999) FEBS Lett. 461, 13-18), potato (Veramendi et al.(1999) Plant Physiol. 121-134), Brassica napus (Accession No. A1352726),Capsicum annum (Accession No. AA840716), Arabidopsis (Accession No.U28215), other sequences are easy to identify by homology comparisons sothat an inhibition of the hexokinase is possible is all relevant cropplants.

[0031] In addition to sense and anti-sense constructs, inhibitors of theappropriate proteins could also be used. Examples for this would be theoverexpression of invertase inhibitors (Greiner et al. (1998) PlantPhysiol. 116, 733-742) or of antibodies which are directed againstparticular proteins. Examples of the successful expression of antibodiesin plants are summarised by Whitelam and Cockburn (Trends in PlantScience (1996), 8, 268-272) and other examples can be obtained from theliterature in the art.

[0032] Other approaches involve controlling the sucrose isomeraseactivity. As described above, the sucrose isomerase activity results inthe formation of palatinose which leads to an undersupply of therelevant cells with carbohydrates. In order to avoid losses of growth,the sucrose isomerase will therefore be expressed preferablycell-specifically in the target cells. Alternatively the sucroseisomerase activity can be controlled by the expression of inhibitors.Inhibitors have been developed in nature for enzymes comparable withsucrose isomerase. An example has already been mentioned, the invertaseinhibitors. Other examples are: proteinase inhibitors (e.g. in Gruden etal. (1997) Plant Mol. Biol. 34, 317-323), polygalacturonase inhibitors(e.g. in Mahalingam et al. (1999) Plant Microb. Interact. 12, 490-498)and amylase inhibitors (e.g. in Grossi et al. (1997) Planta 203,295-303). All these inhibitors bind to the target protein and preventits catalytic activity. Furthermore, the sucrose isomerase could becontrolled by antibodies which bind to the isomerase and thus switch offits activity where desired.

[0033] Finally, for the implementation of the present invention onlysuitable regulatory sequences are required which provide for theexpression of an operatively linked sucrose isomerase DNA sequence inthe anthers, in the tapetum and/or in the pollen of the transformedplants. Here also the person skilled in the art can easily obtainsuitable sequences from the prior art. Some promoter sequencesespecially suitable for the anther- or pollen-specific expression ofcoding sequences are described below.

[0034] These tissue- or cell-specific promoters are also preferably usedfor anti-sense or sense constructs to restrict modifications of thecarbohydrate metabolism with the aim of achieving an undersupply ofpollen also to the relevant tissue.

[0035] Finally, the production of chimeric gene constructs in whichsucrose isomerase coding DNA sequences are under the control ofregulatory sequences, which ensure an anther/tapetum/pollen-specificexpression, is carried out by means of conventional cloning methods(see, for example Sambrook et al. (1989), supra).

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 depicts the cloning of the amplified sucrose isomerasefragment into the vector pCR-Blunt (Invitrogen) to obtain plasmidspCR-SucIso1 (with translation start; SEQ ID NO:12) or pCR-SucIso2(without translation start; SEQ ID NO:10).

[0037]FIG. 2 depicts plasmid pCR-PalQ which was constructed by thecloning of a palatinase sequence from E. rhapontici (fragment A, whichextends from nucleotide 2-1656 of the palatinase gene (see SEQ ID NO:1)) into the vector pCR-Blunt (Invitrogen).

[0038]FIG. 3 depicts plasmid p35S-cwIso (35S=35S promoter, cw=cell wall,Iso=sucrose isomerase). A DNA sequence that codes for a sucroseisomerase was isolated from the plasmid pCR SucIso2 (FIG. 1) bydigestion with BamHI and SalI and ligated in a BamHI/SalI linearised pMAvector. Fragment A contains the 35S promoter of the Cauliflower MosaicVirus (CaMV). Fragment B contains a proteinase inhibitor II gene frompotato which is fused via a linker with the sequence ACC GAA TTG GG (SEQID NO:16) to the sucrose isomerase gene from Erwinia rhapontici, whichcomprises the nucleotides 109-1803. Fragment C contains thepolyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5.

[0039]FIG. 4 depicts plasmid pMAL-SucIso. A DNA sequence that codes fora sucrose isomerase was isolated from the plasmid pCR-SucIso2 (FIG. 1)via restriction enzymes BamHI and SalI and ligated in a BamHI/SalIlinearised pMAL-c2 vector (New England Biolabs). Fragment A contains atac-promoter that allows IPTG-inducible gene expression. Fragment Bcontains a region of the malE gene and the initiation of translation.Fragment C contains the coding region of the sucrose isomerase. FragmentD contains the rrnB-terminator from E. coli.

[0040]FIG. 5. The DNA sequence which codes for a palatinase was fused toa leader peptide of a plant gene necessary for the transport into theendoplasmic reticulum (proteinase-inhibitor II gene from potato) and wasbrought under the control of the promoter from the TA29 gene in tobacco(resulting in plasmid pTA29-cwPalQ (TA29=promoter of the TA29 gene fromtobacco, cw=cell wall, PalQ-palatinase) or under control of the 35S RNApromoter (resulting in plasmid p35S-cwpalQ (35S=35S RNA promoter of theCaMV, cw=cell wall, PalQ=palatinase). Fragment A contains the TA29promoter from Nicotiana tabacum in plasmid pTA29-cwPalQ or the 35S RNApromoter of the Cauliflower Mosaic Virus in plasmid p35S-cwPalQ.Fragment B contains the nucleotides 923-1059 of a proteinase inhibitorII gene from potato which are fused via a linker with the sequence ACCGAA TTG GG (SEQ ID NO:16) to the palatinase gene from Erwiniarhapontici, which comprises the nucleotides 2-1656. Fragment C containsthe polyadenylation signal of gene 3 of the T-DNA of the Ti plasmidpTiACH5), nucleotides 11749-11939. The fragments were cloned intoplasmid pBIN19.

[0041]FIG. 6 depicts plasmid pCR-PalZ. Fragment A, which contains thesequence of the gene encoding trehalulase from E. rhapontici (fromnucleotide 4-1659), was cloned into vector pCR-Blunt (Invitrogen).

DETAILED DESCRIPTION

[0042] The present invention thus relates to a recombinant nucleic acidmolecule comprising

[0043] a) regulatory sequences of a promoter active in anthers, in thetapetum and/or in pollen;

[0044] b) operatively linked thereto a DNA sequence which encodes aprotein having the enzymatic activity of a sucrose isomerase; and

[0045] c) operatively linked thereto regulatory sequences which canserve as transcription, termination and/or polyadenylation signals inplant cells.

[0046] In connection with the present invention, a protein having theenzymatic activity of a sucrose isomerase is understood as a proteinwhich catalyses the isomerisation of sucrose to other disaccharides,wherein the α1 →β2 glycosidic bond between glucose and fructose in thesucrose is converted into another glycosidic bond between twomonosaccharide units, especially into an α1→β6 bond and/or an a1→α1bond. Especially preferably a protein having the enzymatic activity of asucrose isomerase will be understood as a protein being capable ofisomerising sucrose to palatinose and/or trehalulose. In this case, theproportion of palatinose and trehalulose among the total disaccharidesformed by isomerisation of sucrose is ≧2%, preferably ≧20%, morepreferably ≧50% and most preferably ≧60%.

[0047] The DNA sequence, which encodes a protein having the enzymaticactivity of a sucrose isomerase can be isolated from natural sources orsynthesised by known methods. It is possible to prepare or producedesired constructs for the transformation of plants by means of currentmolecular biological techniques (see for example, Sambrook et al.(1989), supra). The cloning, mutagenisation, sequence analysis,restriction analysis and other biochemical and molecular biologicalmethods usually used for gene technological manipulation in prokaryoticcells are well known to the person skilled in the art. Thus, it is notonly possible to produce suitable chimeric gene constructs with thedesired fusion of promoter and sucrose isomerase DNA sequence, butrather the person skilled in the art can, if desired, introduce varioustypes of mutations into the sucrose isomerase coding DNA sequence, whichresults in the synthesis of proteins possibly having modified biologicalproperties. By this means it is firstly possible to produce deletionmutants with which the synthesis of suitably truncated proteins can beachieved by progressive deletion from the 5′ or 3′ end of the coding DNAsequence. Further, it is possible to specifically produce enzymes, whichare localised in specific compartments of the plant cell due to additionof suitable signal sequences. The introduction of point mutations isalso very likely at positions where a modification of the amino acidsequence has an influence, for example, on the enzyme activity or theenzyme regulation. In this way it is possible to produce for examplemutants that are no longer subject to the regulation mechanisms normallyprevailing in the cell via allosteric regulation or covalentmodification. Furthermore, mutants having a modified substrate- orproduct specificity can be produced. Further, mutants having a modifiedactivity-, temperature- and/or pH-profile can be produced. Theproduction of mutants, which have the aim to modify the enzymaticactivity, preferably to yield an increase of the sucrose affinity byreducing the Km value is preferred.

[0048] In a preferred embodiment the DNA sequence, which codes for aprotein having the enzymatic activity of a sucrose isomerase is selectedfrom the group consisting of

[0049] a) DNA sequences comprising a nucleotide sequence which encodethe amino acid sequence given in SEQ ID NO. 6 or fragments thereof,

[0050] b) DNA sequences which comprise the nucleotide sequence given inSEQ ID NO. 4 or parts thereof,

[0051] c) DNA sequences comprising a nucleotide sequence whichhybridises with a complementary strand of the nucleotide sequence of a)or b), or parts of this nucleotide sequence,

[0052] d) DNA sequences comprising a nucleotide sequence which isdegenerate to a nucleotide sequence of c), or parts of this nucleotidesequence,

[0053] e) DNA sequences which represent a derivative, analogue orfragment of a nucleotide sequence of a), b), c) or d).

[0054] Apart from the sucrose isomerase sequence from Erwinia rhaponticigiven in SEQ ID NO. 4, those having a particularly high affinity tosucrose, i.e. corresponding to a low Km value, are used as preferred DNAsequences, e.g. the sucrose isomerase from Pseudomonas mesacidophila (Kmfor sucrose 19.2 mM, Nagai et al. (1994) Biosci. Biotech. Biochem.58:1789-1793) or Serratia plymuthica (Km for sucrose 63.5 mM; McAllisteret al. (1990) Biotechnol. Lett. 12:667-672).

[0055] Within the framework of this invention the term “hybridisation”means hybridisation under conventional hybridisation conditions,preferably under stringent conditions, as described for example, inSambrook et al. (1989, supra).

[0056] DNA sequences which hybridise with DNA sequences coding for aprotein having the enzymatic activity of a sucrose isomerase may, forexample be isolated from genomic or cDNA libraries. Such DNA sequencescan be identified and isolated, for example, by using DNA sequenceswhich exactly or substantially have one of the afore-mentioned sucroseisomerase coding nucleotide sequences of the prior art or parts of thesesequences or the reverse complements of these DNA sequences, e.g. byhybridisation according to standard methods (see, for example, Sambrooket al. (1989), supra). Fragments used as a hybridisation probe can alsobe synthetic fragments produced using conventional synthesis techniquesand whose sequence is substantially identical to one of theafore-mentioned DNA sequences for sucrose isomerase or a part of one ofthese sequences. The DNA sequences, which encode a protein having theenzymatic activity of a sucrose isomerase also comprise DNA sequenceswhose nucleotide sequences are degenerate to one of the DNA sequences asdescribed above. The degeneration of the genetic code offers one skilledin the art, among other things, the possibility of adapting thenucleotide sequence of the DNA sequence to the codon preference (codonusage) of the target plant, i.e. the male sterile plant as a result ofthe specific expression of the sucrose isomerase DNA sequence, andthereby optimising the expression.

[0057] The above-mentioned DNA sequences also comprise fragments,derivatives and allelic variants of the DNA sequences as described abovewhich code for a protein having the enzymatic activity of a sucroseisomerase. “Fragments” are to be understood as parts of the DNA sequencethat are long enough to encode one of the proteins described. The term“derivative” in this context means that the sequences differ from theDNA sequences described above at one or several position/s but have ahigh degree in homology to these sequences. Homology means herein asequence identity of at least 40 percent, especially an identity of atleast 60 percent, preferably more than 80 percent and more preferablymore than 90 percent. The variations to the above described DNAsequences may be caused for example by deletion, substitution, insertionor recombination.

[0058] The variations to the above mentioned DNA sequences can be causedfor example by deletion, substitution, insertion or recombination.

[0059] The DNA sequences that are homologous to the above-mentionedsequences and represent derivatives of these sequences are generallyvariations of these sequences, which represent modifications having thesame biological function. These variations can be both naturallyoccurring variations, for example sequences from other organisms, ormutations, wherein these mutations can have occurred naturally or havebeen introduced by targeted mutagenesis. Moreover, the variations canfurther comprise synthetic sequences. The allelic variants can benaturally occurring and synthetic variants or variants created byrecombinant DNA techniques.

[0060] In a more preferred embodiment the described DNA sequence codingfor a sucrose isomerase originates from Erwinia rhapontici (as given inSEQ ID No. 4).

[0061] The present invention also relates to a recombinant nucleic acidmolecule comprising

[0062] a) regulatory sequences of a promoter active in anthers, in thetapetum and/or in pollen;

[0063] b) a DNA sequence linked thereto in sense or anti-senseorientation, whose transcription results in an inhibition of the plant'sinnate invertase, hexose transporter, hexokinase and/or proton ATPaseexpression, and

[0064] c) operatively linked thereto regulatory sequences which canserve as transcription, termination and/or polyadenylation signals inplant cells.

[0065] For the expression of the DNA sequence contained in therecombinant DNA molecules according to the invention in plant cells, theDNA sequence is linked to regulatory sequences, which ensure thetranscription in plant cells. Any promoter active in plant cells comesinto consideration here. Since according to the invention the sucroseisomerase must be expressed in anther, tapetum and/or pollen tissue, anypromoter, which ensures the expression in anthers, tapetum or pollen,whether it is inter alia in anthers, tapetum or pollen or exclusively inthese tissues, comes into consideration here.

[0066] The promoter can be selected so that the expression takes placeconstitutively or only in anther-, tapetum- and/or pollen-specifictissue, at a particular time in the plant development and/or at a timedetermined by external influences, biotic or abiotic stimuli (inducedgene expression). With reference to the plant to be transformed, thepromoter can be homologous or heterologous. When a constitutive promoteris used, a cell- or tissue-specific expression can also be achieved byinhibiting the gene expression in the cells or tissues in which it isnot desired, for example, by the expression of antibodies that bind tothe gene product and thus suppress its enzymatic activity, or bysuitable inhibitors.

[0067] Particularly suited promoters within the teaching of theinvention are anther-, tapetum- and/or pollen-specific promoters.Examples of this are:

[0068] the promoter of the tap 1 gene from Antirrhinum majus (Sommer etal. (1990) EMBO J. 9:605-613; Sommer et al. (1991) Development, Suppl.1: 169-176; e.g. as 2,2 kb EcoRI/BamHI-restriction fragment);

[0069] the promoter of the TA29 gene (Mariani et al. (1990) Nature347:737-741; Seurinck et al. (1990) Nucl. Acids Res. 18:3403; Gene bankAccession No. X52283);

[0070] the promoter of the RA8 gene from Oryza sativa L. (Jeon et al.(1999) Plant Mol. Biol. 39:35-44, this publication describes expressionstudies with RA8 promoter/GUS constructs in transgenic rice plants);

[0071] the promoter of the Bp 19 gene from Brassica napus (Albani et al.(1991) Plant Mol. Biol. 16:501-513);

[0072] the promoters of the LAT52 und LAT56 gene from tomato (Twell etal. (1990) Development 109:705-713);

[0073] the promoter of the BNA215-6 gene from Brassica campestris L.ssp. Pekinensis (Kim et al. (1997) Mol. Cells 7:21-27, promoter/GUSexpression analyses in transgenic tobacco plants are described here);

[0074] the promoter of the NeIF-4A8 gene from Nicotiana tabacum (Branderand Kuhlemeier (1995) Plant Mol. Biol. 27:637-649, promoter/GUSexpression studies are described here);

[0075] the promoter of the Bgp1 gene from Brassica campestris (Xu et al.(1993) Mol. Gen. Genet. 239:58-65, deletion constructs and theiranalysis in transgenic Arabidopsis thaliana plants are described here);

[0076] the promoter of the APG gene from A. thaliana (Roberts et al.(1993) Plant J. 3:111-120;

[0077] the promoter of the tap2 gene from snapdragon (Nacken et al.(1991) FEBS Lett. 280:155-158, which describes the molecular analysis ofthis anther-specific gene);

[0078] the promoters of the chiA and chiB gene from petunia (van Tunenet al. (1990) Plant Cell 2:393-401);

[0079] the pollen-specific promoters of the Bnm 1 gene from Brassicanapus (Treacy et al. (1997) Plant Mol. Biol. 34:603-611, promoter/GUSexpression analyses in transgenic rape plants are described in thisarticle);

[0080] the promoter of the Bp4 gene from Brassica napus and the promoterof the NTM9 gene from Nicotiana tabacum (Custers et al. (1997) PlantMol. Biol. 35:689-699) are active promoters at the early stages ofpollen development; here a male sterile phenotype could be generated bymeans of promoter/barnase fusion constructs in transgenic tobaccoplants;

[0081] the pollen-specific promoter of the ZM13 gene from maize(Hamilton et al. (1998) Plant Mol. Biol. 38:663-669), the sequenceranges required for pollen-specific expression were identified bymutation analyses and described;

[0082] the pollen-specific promoter of an invertase from potato (Machrayet al. (1999) “The role of invertases in plant carbohydrate partitioningand beyond”, Abstracts, Workshop University of Regensburg, Oct. 3-6,1999; promoter/GUS expression studies are described here).

[0083] The person skilled in the art can obtain other anther-specificgenes or promoters from the prior art, especially from the relevantscientific journals and gene databases. In addition, the person skilledin the art is capable of isolating other suitable promoters by routinemethods. Thus, the person skilled in the art can identifyanther-specific regulatory nucleic acid elements using current molecularbiological methods, for example, hybridisation experiments or DNAprotein binding studies. In this case, as a first step, for example,total poly(A)⁺ RNA is isolated from the anther tissue of the desiredorganism from which the regulatory sequence is to be isolated, and acDNA library is made. As a second step, cDNA clones based on poly(A)⁺RNA molecules from a non-anther tissue are used to identify those clonesfrom the first library by means of hybridisation whose correspondingpoly(A)⁺ RNA molecules only accumulate in anther tissue. Then, thesethus identified cDNAs are used to isolate promoters which haveanther-specific regulatory elements. Other PCR-based methods forisolating suitable anther-specific promoters are also available to theperson skilled in the art. The same applies, of course, also to pollen-or tapetum-specific promoters.

[0084] In a preferred embodiment the anther-specific promoter is theTA29 promoter from tobacco.

[0085] Also present are transcription or termination sequences thatprovide for correct transcription termination and can provide foraddition of a poly(A) tail to the transcript to which a function in thestabilisation of transcripts is assigned. Such elements are described inthe literature and are interchangeable in any order.

[0086] The invention further relates to vectors and micro-organismswhich contain the nucleic acid molecules according to the invention andwhose usage makes it possible to produce male sterile plants. Thevectors especially include plasmids, cosmids, viruses, bacteriophagesand other vectors common in gene technology. The micro-organisms areprimarily bacteria, viruses, fungi, yeasts and algae.

[0087] The invention also relates to a method for producing male sterileplants comprising the following steps:

[0088] a) Production of a recombinant nucleic acid molecule thatcomprises the following sequences:

[0089] regulatory sequences of a promoter active in anthers, in thetapetum and/or in pollen;

[0090] operatively linked thereto a DNA sequence which codes for aprotein having the enzymatic activity of a sucrose isomerase; and

[0091] operatively linked thereto regulatory sequences which can serveas transcription, termination and/or polyadenylation signals in plantcells;

[0092] b) Transfer of the nucleic acid molecule from a) to plant cellsand

[0093] c) Regeneration of transgenic plants.

[0094] The invention further relates to a method for producing malesterile plants comprising the following steps:

[0095] a) Production of a recombinant nucleic acid molecule thatcomprises the following sequences:

[0096] regulatory sequences of a promoter active in anthers, in thetapetum and/or in pollen;

[0097] a DNA sequence linked thereto in sense or anti-sense orientation,whose transcription results in an inhibition of the plant's innateinvertase, hexose transporter, hexokinase and/or proton ATPaseexpression, and

[0098] operatively linked thereto regulatory sequences which can serveas transcription, termination and/or polyadenylation signals in plantcells;

[0099] b) Transfer of the nucleic acid molecule from a) to plant cellsand

[0100] c) Regeneration of transgenic plants.

[0101] The invention also relates to plant cells, which contain thenucleic acid molecules according to the invention, which code for aprotein having the enzymatic activity of a sucrose isomerase. Theinvention also relates to harvest products and propagation material oftransgenic plants as well as to the transgenic plants themselves, whichcontain a nucleic acid molecule according to the invention. Thetransgenic plants of the invention are male sterile as a result of theintroduction and expression of a DNA sequence coding for a sucroseisomerase in the anthers.

[0102] All statements made herein with reference to recombinant nucleicacid molecules which encode a protein having the activity of a sucroseisomerase, whether it is in connection with the production of vectors,plant cells, host cells, transgenic plants and the like also apply tothe other approaches described above for modifying the sucrosemetabolism with the effect of undersupplying the developing pollen withcarbohydrates.

[0103] In order to prepare the introduction of foreign genes into higherplants or their cells a large number of cloning vectors are available,which contain a replicating signal for E. Coli and a marker gene forselecting transformed bacterial cells. Examples of such vectors arepBR322, pUC series, M13 mp series, pACYC184 and the like. The desiredsequence can be introduced into the vector at a suitable restrictionsite. The resulting plasmid is then used for the transformation of E.coli cells. Transformed E. coli cells are cultivated in a suitablemedium and then harvested and lysed, and the plasmid is recovered.Restriction analyses, gel electrophoresis and otherbiochemical-molecular biological methods are generally used as analyticmethods to characterise the plasmid DNA so obtained. After eachmanipulation the plasmid DNA can be cleaved and the thus obtained DNAfragments can be linked to other DNA sequences.

[0104] A plurality of techniques is available for introducing DNA into aplant host cell, wherein the person skilled in the art will not have anydifficulties in selecting a suitable method in each case. As alreadymentioned, these techniques comprise the transformation of plant cellswith T-DNA by use of Agrobacterium tumefaciens or Agrobacteriumrhizogenes as the transforming agent, the fusion of protoplasts, theinjection, electroporation, the direct gene transfer of isolated DNAinto protoplasts, the introduction of DNA by means of biolistic methodsas well as other possibilities which have been well-established forseveral years and belong to the normal repertoire of the person skilledin the art in plant molecular biology or plant biotechnology.

[0105] For the injection and electroporation of DNA into plant cells, nospecial requirements are imposed per se on the plasmids used. The sameapplies to direct gene transfer. Simple 20 plasmids such as pUCderivatives can be used for example. However, if entire plants are to beregenerated from these transformed cells, the presence of a selectablemarker gene is recommended. The person skilled in the art is familiar tothe current selection markers and there is no problem for him to selecta suitable marker.

[0106] Depending on the method for introducing the desired gene into theplant cell, other DNA sequences may be required. If for example the Tior Ri plasmid is used for the transformation of the plant cell, at leastthe right border, however more often both the right and left border ofthe T-DNA in the Ti or Ri plasmid, respectively, must be linked to thegenes to be integrated as a flanking region. If agrobacteria are usedfor the transformation, the DNA to be integrated must be cloned intospecial plasmids and specifically either into an intermediate or into abinary vector. The intermediate vectors can be integrated into the Ti orRi plasmid of the agrobacteria by homologous recombination due tosequences that are homologous to sequences in the T-DNA. This alsocontains the vir-region, which is required for T-DNA transfer.Intermediate vectors cannot replicate in agrobacteria. The intermediatevector can be transferred to Agrobacterium tumefaciens by means of ahelper plasmid (conjugation). Binary vectors are able to replicate in E.coli as well as in agrobacteria. They contain a selection marker geneand a linker or polylinker framed by the right and left T-DNA borderregion. They can be transformed directly into the agrobacteria. Theagrobacterial host cell should contain a plasmid carrying a vir-region.The vir-region is required for the transfer of the T-DNA into the plantcell. Additional T-DNA can be present. Such a transformed agrobacterialcell is used for the transformation of plant cells. The use of T-DNA forthe transformation of plant cells has been studied intensively and hasbeen sufficiently described in generally known reviews and planttransformation manuals. Plant explants can be specifically cultivatedwith Agrobacterium tumefaciens or Agrobacterium rhizogenes for thetransfer of DNA into the plant cell. From the infected plant material(e.g. leaf pieces, stem segments, roots but also protoplasts orsuspension-cultivated plant cells) whole plants may be regenerated againin a suitable medium that can contain antibiotics or biocides to selectthe transformed cells.

[0107] Once the introduced DNA has been integrated into the plant cellgenome, it is generally stable there and is maintained in the progeny ofthe originally transformed cell as well. It normally contains aselection marker, which imparts the transformed plant cells resistanceto a biocide or an antibiotic such as kanamycin, G 418, bleomycin,hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea,gentamycin or phosphinotricin and others. The individually selectedmarker should thus allow the selection of transformed cells from cellslacking the introduced DNA. Alternative markers are also suited for thispurpose such as nutritive markers, screening markers (such as GFP, greenfluorescent protein). Naturally, it could also be done without anyselection marker, although this would involve a fairly high screeningexpenditure. If marker-free transgenic plants are desired, there arestrategies available to the person skilled in the art, which allowsubsequent removal of the marker gene, by e.g. cotransformation,sequence-specific recombinases.

[0108] Transgenic plants are regenerated from transgenic plant cells byusual regeneration methods using known media. By the use of normalmethods, including molecular biological methods such as PCR, blotanalyses, the plants thus obtained may then be analysed for the presenceof introduced DNA which encodes a protein having the enzymatic activityof a sucrose isomerase.

[0109] The transgenic plant or the transgenic plant cells, respectively,can be any monocotyledonous or dicotyledonous plant or plant cell;preferably they are crop plants or cells of crop plants. More preferablythese can be rape, cereals, sugar beet, maize, sunflower and soybean. Inprinciple, however, any crop plant for which hybrid systems areespecially useful and valuable is worthwhile for the implementation ofthe invention.

[0110] The invention also relates to propagation material and harvestproducts of plants according to the invention, for example fruits,seeds, tubers, rhizomes (rootstocks), seedlings, cuttings and the like.

[0111] The transformed cells grow within the plant in the usual way. Theresulting plants can be cultivated normally. The plants differ in theirphenotype from wild-type plants by the male sterile phenotype.

[0112] The specific expression of sucrose isomerase in the anthers ofplants according to the invention or in plant cells according to theinvention can be demonstrated and followed using conventional molecularbiological and biochemical methods. These techniques are known to theperson skilled in the art and he can easily select a suitable method ofdetection, for example a northern blot analysis to detect sucroseisomerase-specific RNA or to determine the level of accumulation ofsucrose isomerase-specific RNA, a southern blot analysis to identify DNAsequences coding for sucrose isomerase or a western blot analysis todetect the sucrose isomerase protein encoded by the DNA sequencesaccording to the present invention. Naturally, the person skilled in theart can, of course, also determine the detection of the enzymaticactivity of sucrose isomerase using protocols available in theliterature.

[0113] As mentioned initially, in addition to a system for producingmale sterility in plants, it is also desirable to have a correspondingrestorer system. In the case where male sterility is produced byanther-specific expression of DNA sequences, which encode a proteinhaving the enzymatic activity of a sucrose isomerase, the male fertilitycan be restored as follows.

[0114] On the one hand, it is possible to use DNA sequences, which codefor a protein having the enzymatic activity of a palatinase as restorergene. The palatinase also known as palatinose hydrolase catalyses thecleavage of the disaccharide palatinose into the hexoses fructose andglucose. On the other hand, alternatively or additionally to the DNAsequences coding for a palatinase, nucleic acid sequences which code fora protein having the enzymatic activity of a trehalulase can be used asrestorer genes. Trehalulase, also known as trehalulose hydrolase,catalyses the cleavage of the disaccharide trehalulose also intofructose and glucose.

[0115] Thus, the male sterile phenotype can be overcome or neutralisedby crossing with plants, which express a protein having the enzymaticactivity of a palatinase and/or a protein having the enzymatic activityof a trehalulase, and thus a complete hybrid system including restorerfunction can be realised.

[0116] Palatinase genes are known in the prior art. Thus, PCT/EP95/00165 discloses the sequence of a palatinase gene from the bacteriumProtaminobacter rubrum and the sequence of a palatinase gene from thebacterium Pseudomonas mesoacidophila MX-45.

[0117] Now disclosed for the first time as part of the present inventionis a DNA sequence from Erwinia rhapontici, which codes for a proteinhaving the enzymatic activity of a palatinase. This sequence is given inthe appended sequence protocol in SEQ ID No. 1, and the derived aminoacid sequence is given in SEQ ID NOs: 2 and 3.

[0118] Also provided for the first time, as part of the presentinvention, is a DNA sequence from Erwinia rhapontici, which encodes aprotein having the enzymatic activity of a trehalulase. The sequence isgiven in the appended sequence protocol in SEQ ID NO: 7, and the derivedamino acid sequence is given in SEQ ID NOS: 8 and 9.

[0119] In connection with palatinase and trehalulase sequences fromother sources and the methods by which the person skilled in the art canisolate or produce such sequences, reference is made to the reasoningput forward above in connection with sucrose isomerase sequences intheir full extent. The same applies to the production of recombinantnucleic acid molecules which code for a protein having the enzymaticactivity of a palatinase or a protein having the enzymatic activity of atrehalulase for the production of plants and also for the transfer ofsuch nucleic acid molecules to plant cells and the regeneration oftransgenic plants. Reference is also expressly made to all the abovereasoning on the hybridisation, homology, derivatives, variants andfragments, regulatory sequences etc.

[0120] The invention thus also relates to the nucleotide sequences givenin SEQ ID NO: 1 and SEQ ID NO: 7, respectively, which encode a proteinhaving the enzymatic activity of a palatinase or trehalulase and the useof nucleic acid molecules, which encode proteins having the enzymaticactivity of a palatinase or trehalulase for the restoration of malefertility in transgenic plants.

[0121] The invention further relates to a method for the production ofmale fertile hybrid plants comprising the following steps:

[0122] a) Production of a first transgenic male sterile parent plantcomprising a nucleic acid molecule which codes for a protein having theenzymatic activity of a sucrose isomerase,

[0123] b) Production of a second transgenic parent plant, comprising anucleic acid molecule which encodes a protein having the enzymaticactivity of a palatinase and/or a nucleic acid molecule which codes fora protein having the enzymatic activity of a trehalulase,

[0124] c) Crossing the first parent plant with the second parent plantto produce a hybrid plant, wherein the hybrid plant is male fertile.

[0125] The same promoters as are useful for the expression of thesucrose isomerase are, of course, also suitable for the expression ofthe palatinase or trehalulase gene. The palatinase or trehalulase DNAsequences can advantageously also be expressed under the control ofconstitutive promoters, such as for example the 35S RNA promoter ofCaMV. Both the palatinase and the trehalulase enzyme activity have perse no influence on the plant cells and thus no influence on plantgrowth, even when expressed in all tissues of the transgenic plant.

[0126] Preferably used are those palatinase DNA sequences, which codefor an enzyme with high affinity to palatinose. Accordingly, preferablyused are those trehalulase DNA sequences, which code for an enzyme withhigh affinity to trehalulose.

[0127] As mentioned above, the provision of a restorer system, that isthe re-establishment of the fertility of a transgenic male sterileplant, requires the production of a second, so-called restorer line oftransgenic plants. This restorer line can thus be a line, which containsa DNA sequence that codes for a protein having the activity of apalatinase or a trehalulase. Other approaches to the restoration offertility are also possible. Thus, the expression of a correspondingsucrose isomerase inhibitor in the restorer line can be used to restorefertility in sucrose isomerase-expressing male sterile plants. Such aninhibitor can, for example, be an antibody directed towards the sucroseisomerase or an inhibitor, as it is known for invertases, for example(Greiner et al. (1999) Nat. Biotechnol. 17:708-711).

[0128] In another approach, the restorer line can express a ribozymedirected towards the sucrose isomerase mRNA. Ribozymes can be producedin such a fashion that they possess endonuclease activity directedtowards a specific mRNA (see, for example, Steinecke et al. (1992) EMBOJ. 11: 1525).

[0129] In a similar approach the fertility can be restored by theexpression of a corresponding anti-sense RNA. Binding of the antisenseRNA to the target RNA results in inhibition of its translation (Patersonet al. (1987) Proc. Natl. Acad. Sci. USA 74:4370). In connection withthe present invention, one would thus crossbred a plant which is malesterile due to the expression of the sucrose isomerase with a restorerline in which the corresponding sucrose isomerase sequences are underthe control of suitable promoters in the anti-sense orientation so thatanti-sense transcripts for sucrose isomerase are formed. By this means,the anther-specific sense expression, which brings about the malesterile phenotype is inhibited or neutralised so that male fertilecrossing products are formed. Alternatively, the phenomenon ofcosuppression can be used in the same way as the anti-sense technique torestore male fertility. In a preferred embodiment of the invention, theexpression of the anti-sense or cosuppression RNA is under the controlof an inducible promoter whose activation allows the specificrestoration of the male fertility.

[0130] Another alternative includes the expression of a RNA transcript,which causes the RNAse-P-mediated cleavage of sucrose isomerase mRNAmolecules. In this approach an external leader sequence is constructedwhich directs the endogenous RNAse-P to sucrose isomerase mRNA andfinally mediates the cleavage of this mRNA (Altman et al., U.S. Pat. No.5,168,053; Yuan et al. (1994) Science 263:1269). Preferably the externalleader sequence includes 10 to 15 nucleotides complementary to sucroseisomerase and a 3′-NCCA nucleotide sequence wherein N is preferably apurine. The transcripts of the external leader sequence bind to thetarget mRNA via base pairing which facilitates the cleavage of the mRNAby the RNAse-P at the nucleotide 5′ from the base paired region.

[0131] Another approach to the restoration of male fertility aretransgenic, male sterile plants which, in addition to a sucroseisomerase gene operatively linked to a promoter sequence, contain aprokaryotic control region within the same expression cassette.Transgenic male fertile plants, which express a prokaryotic polypeptideunder the control of a suitable promoter, are additionally produced. Inthe F1 hybrids the prokaryotic polypeptide binds to the prokaryoticcontrol region and represses the expression of the sucrose isomerase.Specifically, the LexA gene/LexA operator system can be used to controlthe gene expression (U.S. Pat. No. 4,833,080; Wang et al. (1993) Mol.Cell Biol. 13:1805). This would mean that the expression cassette of themale sterile line contains the LexA operator sequence whereas theexpression cassette of the male fertile restorer line contains thecoding region of the LexA repressor. In the F1 hybrids the LexArepressor binds to the LexA operator region and thereby preventstranscription of the sucrose isomerase gene. LexA operator-DNA moleculescan be obtained, for example by the synthesis of DNA fragments, whichcontain LexA operator sequences well known to the person skilled in theart from the literature, as described, for example, by Garriga et al.(1992) Mol. Gen. Genet. 236:125. DNA sequences which code for the LexArepressor can, for example, be obtained by synthesis of such DNAmolecules or by DNA cloning techniques as are known to the personskilled in the art and are described, for example by Garriga et al.,vide supra. Alternatively, sequences coding for the LexA repressor canbe taken, for example, from plasmid pRB500 (ATTC 67758).

[0132] The approach explained in connection with the LexArepressor/operator system for the re-establishment of male fertility canalso be achieved with other repressor/operator systems as the personskilled in the art knows them from the literature in a plurality ofways, e.g. the Lac repressor/lac operator system or the trprepressor/trp operator system.

[0133] Finally, the fertility can be restored by application of chemicalcompounds or substances during pollen development, which inhibit theactivity of the sucrose isomerase.

[0134] The invention is based on the successful production of new plantswhich are male sterile due to the introduction and expression of anucleic acid sequence coding for a sucrose isomerase in the anthers,which is explained in the following examples which serve merely toillustrate the invention and are in no way to be understood asrestrictive.

EXAMPLES

[0135] Gene technological methods on which the embodiments are based:

[0136] 1. General Cloning Methods

[0137] Cloning methods, such as for example: restriction cleavage, DNAisolation, agarose gel electrophoresis, purification of DNA fragments,transfer of nucleic acids onto nitrocellulose and nylon membranes,linking of DNA fragments, transformation of E. coli cells, cultivationof bacteria, sequence analysis of recombinant DNA, were performed asdescribed in Sambrook et al. (1989, vide supra). The transformation ofAgrobacterium tumefaciens was carried out according to the method ofHöfgen and Willmitzer (1988, Nucl. Acids Res. 16:9877). Agrobacteriawere cultivated in YEB medium (Vervliet et al. (1975) Gen. Virol.26:33).

[0138] 2. Production of a Genomic Library of Erwinia rhapontici

[0139] In order to produce a genomic library from Erwinia rhapontici(DSM 4484) chromosomal DNA was isolated from the cells of a 50 mlovernight culture according to a standard protocol. Approximately 300 μgof the DNA were then partially digested with the restriction enzymeSau3A and separated on a preparative agarose gel. Fragments between 5and 12 kb were eluted from the gel using the Qiaquick Gel Extraction Kit(Qiagen, Hilden). The resulting DNA fragments were ligated inBamHI-digested Lambda ZAP-Express-Arme (Stratagene, La Jolla, USA) andthen packed in vitro (Gigapack III Gold Packaging Extract, Stratagene,according to the manufacturer's data). E. coli bacteria of the strainXL-MRF′ (Stratagene) were then infected with recombinant lambda phages,the titre of the library was determined and the library was thenamplified.

[0140] 3. Screening of a Genomic Library

[0141] Approximately 10⁵ phages were plated for the isolation of genomicclones. After transferring the phages onto nylon filters (Genescreen,NEN) the filters were hybridised with a radioactively labelled DNAfragment. Positive signals were visualised by autoradiography andsingling out was performed.

[0142] 4. Bacterial Strains and Plasmids

[0143]E. coli (XL-1 Blue, XL-MRF′ and XLOLR) bacteria were obtained fromStratagene. Erwinia rhapontici (DSM 4484) was obtained from the DeutscheSammlung für Mikro-organismen und Zellkulturen GmbH (Braunschweig,Germany). The agrobacterial strain used for the transformation of plants(C58C1 with the plasmid pGV 3850kan) was described by Debleare et al.(1985, Nucl. Acids Res. 13:4777). The vectors pCR-Blunt (Invitrogen,Netherlands), pMAL-c2 (New England Biolabs), pUC19 (Yanish-Perron (1985)Gene 33:103-119) and Bin19 (Bevan (1984) Nucl. Acids Res. 12:8711-8720)were used for the cloning.

[0144] 5. Transformation of Tobacco

[0145] For the transformation of tobacco plants (Nicotiana tabacum L.cv. Samsun NN) 10 ml of a overnight culture of Agrobacterium tumefaciensgrown under selection was centrifuged, the supernatant was discarded,and the bacteria were resuspended in the same volume, butantibiotic-free medium. Leaf disks of sterile plants (diameter approx. 1cm) were bathed in this bacteria solution in a sterile culture dish. Theleaf disks were then placed into Petri dishes onto MS medium (Murashigeand Skoog (1962) Physiol. Plant 15:473) containing 2% sucrose and 0.8%Bacto-agar. After incubation for 2 days in darkness at 25° C. they weretransferred to MS medium containing 100 mg/l kanamycin, 500 mg/lclaforan, 1 mg/l benzylaminopurine (BAP), 0.2 mg/l naphthylacetic acid(NAA), 1.6% glucose and 0.8% Bacto-agar and cultivation was continued(16 hours light/8 hours darkness). Growing shoots were transferred to ahormone-free MS medium containing 2% sucrose, 250 mg/l claforan and 0.8%Bacto-agar.

[0146] 6. Detection of Palatinose in Plant Extracts

[0147] In order to detect sucrose isomerase activity in plant extracts,leaf disks having a diameter of approx. 0.8 cm were extracted for 2 h at70° C. using 100 μl 80% ethanol and 10 mM HEPES buffer (pH 7.5). A HPLCsystem from Dionex that was equipped with a PA-1 (4×250 mm) column and apulsed electrochemical detector was used to analyse an aliquot of theseextracts. Prior to injection the samples were centrifuged for 2 minutesat 13,000 rpm. Sugars were then eluted using a gradient of 0 to 1 Msodium acetate for 10 minutes, after 4 minutes at 150 mM NaOH and a flowrate of 1 ml/min. Suitable standards obtained from Sigma were used toidentify and quantify the sugars.

Example 1

[0148] PCR Amplification of a Subfragment of Sucrose Isomerase fromErwinia rhapontici

[0149] A subfragment of sucrose isomerase was cloned by polymerase chainreaction (Polymerase Chain Reaction, PCR). The template material wasgenomic DNA from E. rhapontici (DSM 4484), which was isolated accordingto a standard protocol. The amplification was carried out using thespecific primers FB83 5′-GGATCCGGTACCGTTCAGCAATCAAAT-3′ (SEQ ID NO:10)and FB84 5′-GTCGACGTCTTGCCAAAAACCTT-3′, (SEQ ID NO:11)

[0150] which were derived from a sucrose isomerase sequence of the priorart. Primer FB 83 comprises the bases 109-127 and primer FB 84 comprisesthe bases 1289-1306 of the coding region of the sucrose isomerase genefrom E. rhapontici. The PCR reaction mix (100 μl) contained bacterialchromosomal DNA (1 μg), primers FB 83 and FB 84 (250 ng of each), PfuDNA polymerase reaction buffer (10 μl, Stratagene), 200 μM dNTPs (dATP,dCTP, dGTP, dTTP) and 2.5 units of Pfu DNA polymerase (Stratagene).Prior to the initiation of the amplification cycles the mixture washeated for 5 min to 95° C. The polymerisation steps (30 cycles) werecarried out in an automated T3-Thermocycler (Biometra) according to thefollowing program: denaturation at 95° C. (1 minute), annealing of theprimers at 55° C. (40 seconds), polymerase reaction at 72° C. (2minutes). The resulting fragment was cloned into the vector pCR-Blunt(Invitrogen). The identity of the amplified DNA was verified by sequenceanalysis.

[0151] The amplified subfragment can well be used as a hybridisationprobe for the isolation of further sucrose isomerase DNA sequences fromother organisms or as a probe for the analysis of transgenic cells andplants.

Example 2

[0152] Isolation and Sequencing of the Palatinose Operon from E.rhapontici

[0153] A genomic library was screened with a subfragment of the sucroseisomerase (see Example 1) to isolate the palatinose operon. Hence,several positive clones were isolated. By complete sequencing andlinking of these clones it was possible to identify several open readingframes which code for enzymes of palatinose metabolism (see overview ofthe genes of the palatinose operon and the respective gene products asgiven below). The following draft gives a schematic overview of thecloned palatinose gene cluster from Erwinia rhapontici. Arrows indicatethe position of the open reading frames and the direction oftranscription. Gene Function of the gene product palI sucrose isomerasepalR regulator protein of the LysR family palE palatinose bindingprotein, component of the ABC-transporter system for the uptake ofpalatinose into the cell palF integral membrane protein, permease,component of the ABC- transporter system palG integral membrane protein,permease, component of the ABC- transporter system palH presumablyhydrolase activity palK ATP binding protein, component of theABC-transporter system, provides energy for the uptake of palatinoseinto the cell palQ palatinase palZ trehalulase

Example 3

[0154] PCR Amplification of a Sucrose Isomerase from Erwinia rhapontici

[0155] The entire open reading frame of sucrose isomerase was cloned bymeans of polymerase chain reaction (Polymerase Chain Reaction, PCR). Thetemplate material was genomic DNA from E. rhapontici (DSM 4484), whichwas isolated according to a standard protocol. The amplification wascarried out using the specific primers (SEQ ID NO:12) FB965′-GGATCCACAATGGCAACCGTTCAGCAATCAAAT-3′ and (SEQ ID NO:13) FB975′-GTCGACCTACGTGATTAAGTTTATA-3′.

[0156] for pCR-SucIso1. Primer FB 96 comprises the bases 109-127 andadditionally contains a start codon, primer FB 97 contains the bases1786-1803 of the coding region of the sucrose isomerase gene. FB 83(5′-GGATCCGGTACCGTTCAGCAATCAAAT-3′; SEQ ID NO:10), which contains noadditional ATG, was used as 5′ primer to produce the constructpCR-SucIso2. For cloning the amplified DNA into expression vectors theprimers also contain the following restriction sites: primer FB 96 or FB83, BamHI; primer FB 97, SalI. The PCR reaction mix (100 μl) containedbacterial chromosal DNA (1 μg), primer FB 96 and FB 97 for pCR-SucIso1or primer FB 83 and FB 97 for pCR-SucIso2 (250 ng in each case), Pfu DNApolymerase reaction buffer (10 μl, Stratagene), 200 μM dNTPs (dATP,dCTP, dGTP, dTTP) and 2.5 units of Pfu DNA polymerase (Stratagene).Prior to the initiation of the amplification cycles the mixture washeated for 5 min to 95° C. The polymerisation steps (30 cycles) werecarried out in an automated T3-Thermocycler (Biometra) according to thefollowing program: denaturation at 95° C. (1 minute), annealing of theprimers at 55° C. (40 seconds), polymerase reaction at 72° C. (2minutes). The amplified sucrose isomerase fragment was cloned into thevector pCR-Blunt (Invitrogen) by means of which the plasmid pCR-SucIso1(with translation start) or pCR-SucIso2 (without translation start) wasobtained (see FIG. 1). The identity of the amplified DNA was verified bymeans of sequence analysis.

[0157] The fragment A contains the sequence of a sucrose isomerase fromE rhapontici, which extends from nucleotide 109-1803 of the sucroseisomerase gene. The nucleotide sequence of the primer used wasunderlined in each case. The DNA sequence is given in SEQ ID NO: 4.

Example 4

[0158] PCR Amplification of a Palatinase from Erwinia rhapontici

[0159] The entire open reading frame of the palatinase was cloned usingpolymerase chain reaction (Polymerase Chain Reaction, PCR). The templatematerial was genomic DNA from E. rhapontici, which was isolatedaccording to a standard protocol. The amplification was carried outusing the specific primers: FB180 5′-GAGATCTTGCGCAGCACACCGCACTGG-3′ (SEQID NO:14) FB176 5′-GTCGACTCACAGCCTCTCAATAAG-3′ (SEQ ID NO:15)

[0160] Primer FB 180 comprises the bases 2-21, primer FB 176 comprisesthe bases 1638-1656 of the coding region of the palatinase gene. Forcloning the DNA into expression vectors the primers also have thefollowing restriction sites: primer FB 180 BglII; primer FBI 76 SalI.The PCR reaction mix (100 μl) contained bacterial chromosomal DNA (1μg), primer FBI 80 and FB 176 (250 ng in each case), Pfu DNA polymerasereaction buffer (10 μl, Stratagene), 200 μM dNTPs (dATP, dCTP, dGTP,dTTP) and 2.5 units of Pfu DNA polymerase (Stratagene). Beforeinitiating the amplification cycles, the mixture was heated for 5 min to95° C. The polymerisation steps (30 cycles) were carried out in anautomated T3-Thermocycler (Biometra) according to the following program:denaturation at 95° C. (1 minute), annealing of the primers at 55° C.(40 seconds), polymerase reaction at 72° C. (2 minutes). Thecorresponding fragment was cloned into the vector pCR-Blunt(Invitrogen), resulting in the plasmid pCR-PalQ (FIG. 2). The identityof the amplified DNA was verified by sequence analysis. The fragment Acontains the sequence of a palatinase from E. rhapontici, which extendsfrom nucleotide 2-1656 of the palatinase gene (see SEQ ID NO: 1).

Example 5

[0161] Production of plasmid p35S-cwIso

[0162] A DNA sequence which codes for a sucrose isomerase was isolatedfrom the plasmid pCR-SucIso2 and was linked to the 35S promoter of theCauliflower Mosaic Virus, which mediates a constitutive expression intransgenic plant cells, a leader peptide of a plant gene necessary forthe transport (uptake) into the endoplasmic reticulum(proteinase-inhibitor II gene from potato (Keil et al. (1986) Nucl.Acids Res. 14:5641-5650; Gene bank Accession No. X04118), and a planttermination signal. For this purpose the sucrose isomerase fragment wascut out from the pCR-SucIso2 construct (see FIG. 1) by digestion via therestriction sites BamHI and SalI and ligated in a BamHI/SalI linearisedpMA vector. The vector pMA is a modified form of the vector pBinAR(Höfgen and Willmitzer (1990) Plant Sci. 66:221-230) which contains the35S promoter of the Cauliflower Mosaic Virus, which mediates aconstitutive expression in transgenic plants, a leader peptide of theproteinase inhibitor II from potato which mediates the target control ofthe fusion protein into the cell wall, and a plant termination signal.The plant termination signal contains the 3′ end of the polyadenylationsite of the octopine synthase gene. Between the subsequence of theproteinase inhibitor and the termination signal are specific sites forthe restriction enzymes BamHI, XbaI, SalI, PstI and SphI (in thisorder), which allow the insertion of corresponding DNA fragments so thata fusion protein is created between the proteinase inhibitor and theintroduced protein which is then transported into the cell wall oftransgenic plant cells which express this protein (FIG. 3).

[0163] Fragment A contains the 35S promoter of the Cauliflower MosaicVirus (CaMV). It contains one fragment which comprises the nucleotides6909 or 7437 of the CaMV (Franck (1980) Cell 21:285.

[0164] Fragment B contains the nucleotides 923-1059 of a proteinaseinhibitor II gene from potato (Keil et al., supra), which is fused via alinker with the sequence ACC GAA TTG GG (SEQ ID NO:16) to the sucroseisomerase gene from Erwinia rhapontici, which comprises the nucleotides109-1803. By this means a leader peptide of a plant protein necessaryfor the transport of proteins into the endoplasmic reticulum (ER) isN-terminally fused to the sucrose isomerase sequence.

[0165] Fragment C contains the polyadenylation signal of gene 3 of theT-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3:835),nucleotides 11749-11939.

[0166] In p35S-cwIso (35S=35S promoter, cw=cell wall, Iso=sucroseisomerase) the coding region of the sucrose isomerase from E. rhaponticiis under constitutive control and the gene product is transported intothe ER via uptake.

Example 6

[0167] Production of Plasmid pTA29-cwIso

[0168] In a manner similar to that described in Example 5, the plasmidpTA29-cwIso was produced, but with the variation that the expression ofthe fusion protein from proteinase inhibitor leader peptide and thesucrose isomerase is brought under the control of the anther-specificpromoter TA29 from tobacco. The functionality of the anther-specificTA29 promoter has already been demonstrated (Mariani et al. (1990)Nature 347:727-741). The plant termination signal contains the 3′ end ofthe polyadenylation site of the octopine synthase gene. The plasmidpTA29-cwIso contains three fragments A, B and C, which were cloned intothe restriction sites for restriction enzymes of the polylinker of pUC18(see FIG. 3).

[0169] Fragment A contains the TA29 promoter from Nicotiana tabacum. Thefragment contains the nucleotides −1477 to +57 relative to thetranscription initiation site of the TA29 gene (Seurinck et al. (1990)Nucl. Acids. Res. 18:3403). It was amplified by means of PCR fromgenomic DNA of Nicotiana tabacum Var. Samsun NN. The amplification wascarried out using the specific primers: FB1585′-GAATTCGTTTGACAGCTTATCATCGAT-3′ (SEQ ID NO:17) and FB1595′-GGTACCAGCTAATTTCTTTAAGTAAA-3′. (SEQ ID NO:18)

[0170] For cloning the DNA into the expression cassette the primers alsohave the following restriction sites: primer FB 158, EcoRI; primer FB159, Asp718. The PCR reaction mix (100 μl) contained genomic DNA oftobacco (2 μg), primers FB158 and FB159 (250 ng in each case), Pfu DNApolymerase reaction buffer (10 μl, Stratagene), 200 μM dNTPs (dATP,dCTP, dGTP, dTTP) and 2.5 units of Pfu DNA-polymerase (Stratagene).Before initiating the amplification cycles the mixture was heated for 5min to 95° C. The polymerisation steps (35 cycles) were carried out inan automated T3-Thermocycler (Biometra) according to the followingprogram: denaturation at 95° C. (1 minute), annealing of the primers at55° C. (40 seconds), polymerase reaction at 72° C. (2 minutes). Theamplicon was digested with the restriction enzymes EcoRA and Asp718 andcloned into the corresponding restriction sites of the polylinker ofpUC18. The identity of the amplified DNA was verified by sequenceanalysis.

[0171] Fragment B contains the nucleotides 923 to 1059 of a proteinaseinhibitor II gene from potato (Keil et al. (1986) Nucl. Acids Res.14:5641-5650; Gene bank Accession No. X04118) which are fused via alinker with the sequence ACC GAA TTG GG (SEQ ID NO:16) to the sucroseisomerase gene from E. rhapontici, which comprises the nucleotides 109to 1803. By this means a leader peptide of a plant protein required forthe transport of proteins into the ER is N-terminally fused to thesucrose isomerase sequence. The fragment B was cut out as an Asp718/SalIfragment from the p35S-cwIso construct as described above (Example 5)and cloned between the restriction sites Asp718 and SalI of thepolylinker region of pUC18.

[0172] Fragment C contains the polyadenylation signal of gene 3 of theT-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3:835),nucleotides 11749-11939, which was isolated as a PvuII/HindIII fragmentfrom the plasmid pAGV 40 (Herrera-Estrella et al. (1983) Nature 303:209)and has been cloned after addition of SphI linkers to the PvuII sitebetween the SphI/HindIII-site of the polylinker of pUC18.

[0173] The chimeric gene was then cloned as a EcoRI/HindIII fragmentbetween the EcoRI- and HindIII-site of the plasmid pBIN19 (Bevan (1984)Nucl. Acids Res. 12:8711).

[0174] In pTA29-cwIso (TA29=promoter of the TA29 gene from tobacco,cw=cell wall, Iso=sucrose isomerase) the coding region of the sucroseisomerase gene from E. rhapontici is under anther-specific control, thegene product is transported into ER via uptake.

[0175] Tobacco plant cells were transformed as described above with theconstruct pTA29-cwIso by means of agrobacterium-mediated gene transferand whole tobacco plants were regenerated. The resulting pTA29-cwIsotransformants showed a male sterile phenotype, otherwise there were nodifferences in their phenotype compared to the wild-type.

Example 7

[0176] Production of Plasmid pTA29-cwPalQ

[0177] In a manner similar to that described in Example 6, the codingregion of the palatinase from E. rhapontici was fused to a leaderpeptide of a plant gene necessary for the transport into the ER(proteinase inhibitor II gene from potato, Keil et al. (1986) videsupra) under the control of the anther-specific promoter of the TA29gene from tobacco. The resulting construct pTA29-cwPalQ consists ofthree fragments A, B and C (seen in FIG. 5) and allows the expression ofthe palatinase in the cell wall of tapetum cells.

[0178] Fragment A contains the TA29 promoter from Nicotiana tabacum. Thefragment contains the nucleotides −1477 to +57 relative to thetranscription initiation site of the TA29 gene (Seurinck et al. (1990)Nucl. Acids. Res. 18:3403). It was amplified by means of PCR fromgenomic DNA of Nicotiana tabacum Var. Samsun NN. The amplification wascarried out using the specific primers: FB1585′-GAATTCGTTTGACAGCTTATCATCGAT-3′ (SEQ ID NO:17) and FB1595′-GGTACCAGCTAATTTCTTTAAGTAAA-3′. (SEQ ID NO:18)

[0179] For cloning the DNA into the expression cassette the primers alsohave the following restriction sites: primer FB158, EcoRI; primer FB159,Asp718. The PCR reaction mix (100 μl) contained genomic DNA of tobacco(2 μg), primers FB158 and FB159 (250 ng in each case), Pfu DNApolymerase reaction buffer (10 μl, Stratagene), 200 μM dNTPs (dATP,dCTP, dGTP, dTTP) and 2.5 units of Pfu DNA-polymerase (Stratagene).Prior to the initiation of the amplification cycles the mixture washeated for 5 min to 95° C. The polymerisation steps (35 cycles) werecarried out in an automated T3-Thermocycler (Biometra) according to thefollowing program: denaturation at 95° C. (1 minute), annealing of theprimers at 55° C. (40 seconds), polymerase reaction at 72° C. (2minutes). The amplicon was digested with the restriction enzymes EcoRAand Asp718 and cloned into the corresponding restriction sites of thepolylinker of pUC18. The identity of the amplified DNA was verified bysequence analysis.

[0180] Fragment B contains the nucleotides 923-1059 of a proteinaseinhibitor II gene from potato (Solanum tuberosum, Keil et al. 1986, videsupra) which are fused via a linker with the sequence ACC GAA TTG GG(SEQ ID NO: 16) to the palatinase gene from Erwinia rhapontici, whichcomprises the nucleotides 2-1656. By this means a leader peptide of aplant protein required for the transport of proteins into theendoplasmic reticulum is N-terminally fused to the palatinase sequence.

[0181] For cloning fragment B the region of the proteinase inhibitor IIgene comprising the nucleotides 923 to 1059 was isolated via therestriction enzymes Asp718 and BamHI from the pMA vector and clonedbetween the corresponding sites of the polylinker of pUC18. Finally, thepalatinase fragment cut out from the pCR-PalQ construct via BglII andSalI was fused to the sequence of the proteinase inhibitor via the BamHIsite compatible to the BglII site.

[0182] Fragment C contains the polyadenylation signal of gene 3 of theT-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3:835),nucleotides 11749-11939, which was isolated as a PvuII/HindIII fragmentfrom the plasmid pAGV40 (Herrera-Estrella et al. (1983) Nature 303:209)and has been cloned after adding SphI linkers to the PvuII site betweenthe SphI- and HindIII-sites of the polylinker of pUC18. The chimericgene was then cloned as a EcoRI/HindIII fragment between the EcoRI- andHindIII-sites of the plasmid pBIN19 (Bevan (1984) Nucl. Acids Res.12:8711).

[0183] In pTA29-cwPalQ (TA29=promoter of the TA29 gene from tobacco,cw=cell wall, PalQ−palatinase) the coding region of the palatinase genefrom E. rhapontici is under anther-specific control, the gene product istransported into the ER.

[0184] Transgenic plants, which were transformed with pTA29-cwPalQ bymeans of agrobacterium-mediated gene transfer, showed no difference intheir phenotype compared to the wild-type. The daughter plants obtainedfrom crossing these plants with the male sterile plants from Example 6again showed the male fertile phenotype of the pTA29-cwPalQ parentplants.

Example 8

[0185] Production of plasmid p35S-cwPalQ

[0186] The DNA sequence which codes for a palatinase was fused to aleader peptide of a plant gene necessary for the transport into theendoplasmic reticulum (proteinase-inhibitor II gene from potato (Solanumtuberosum, Keil et al. (1986, vide supra)) and was brought under controlof the 35S RNA promoter, resulting in the constructed plasmidp35S-cwpalQ.

[0187] For this purpose the palatinase fragment was cut out from thepCR-palQ construct via the restriction sites BglII and SalI and ligatedin a BamHI/SalI linearised pMA vector. The vector pMA is a modified formof the vector pBinAR (Höfgen and Willmitzer (1990) Plant Sci.66:221-230) which contains the 35S promoter of the Cauliflower MosaicVirus, which mediates a constitutive expression in transgenic plants, aleader peptide of the proteinase inhibitor II from potato (Keil et al.1986, vide supra) which mediates the target control of the fusionprotein into the cell wall, and a plant termination signal. The planttermination signal contains the 3′ end of the polyadenylation site ofthe octopine synthase gene. Between the partial sequence of theproteinase inhibitor and the termination signal are specific sites forthe restriction enzymes BamHI, XbaI, SalI, PstI and SphI (in thisorder), which allow the insertion of corresponding DNA fragments so thata fusion protein is created between the proteinase inhibitor and theintroduced protein which is then transported into the cell wall oftransgenic plants or plant cells which express this protein (see FIG.5).

[0188] Fragment A contains the 35S RNA promoter of the CauliflowerMosaic Virus (CaMV). It contains one fragment which comprises thenucleotides 6909 to 7437 of the CaMV (Franck (1980) Cell 21:285).

[0189] Fragment B contains the nucleotides 923-1059 of a proteinaseinhibitor II gene from potato (Solanum tuberosum, Keil et al. 1986, videsupra), which is fused via a linker having the sequence ACC GAA TTG GGto the palatinase gene from Erwinia rhapontici, which comprises thenucleotides 2-1656. By this means a leader peptide of a plant proteinnecessary for the transport of proteins into the endoplasmic reticulum(ER) is N-terminally fused to the palatinase sequence.

[0190] Fragment C contains the polyadenylation signal of gene 3 of theT-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3:835),nucleotides 11749-11939.

[0191] In p35S-cwPalQ (35S=35S RNA promoter of the CaMV, cw=cell wall,PalQ=palatinase) the coding region of the palatinase gene from E.rhapontici is under constitutive control and the gene product istransported into the ER.

[0192] Transgenic plants, which were transformed with p35S-cwPal bymeans of agrobacterium-mediated gene transfer, showed no difference intheir phenotype compared to the wild-type. The daughter plants obtainedfrom crossing these plants with the male sterile plants from Example 6again showed the male fertile phenotype of the p35S-cwPal parent plants.

Example 9

[0193] Production of the Plasmid pMAL-SucIso

[0194] To produce the plasmid pMAL-SucIso the sucrose isomerase fragmentwas cut out from the construct pCR-SucIso2 via the restriction enzymesBamHI and SalI and ligated in a pMAL-c2 vector (New England Biolabs),which was also cut in this manner to create the construct pMAL-SucIso(FIG. 4). This allows an expression of the enzyme as fusion protein withthe maltose-binding protein under control of the IPTG-inducibletac-promoter.

[0195] Fragment A contains the tac-promoter that allows IPTG-induciblegene expression.

[0196] Fragment B contains a region of the malE gene and the initiationof translation.

[0197] Fragment C contains the coding region of the sucrose isomerase.

[0198] Fragment D contains the rrnB-terminator from E. coli.

[0199] Bacterial cells transformed with pMAL-SucIso show IPTG-inducibleexpression of the sucrose isomerase from E. rhapontici.

Example 10

[0200] Functional Detection of Sucrose Isomerase Activity in E. coli

[0201] Functional characterisation of the sucrose isomerase gene wasimplemented by expression in E. coli. For this purpose the plasmidpMAL-Suclso was transformed in E. coli (XL-I blue, Stratagene). Theexpression of the fusion protein between the maltose-binding protein andthe sucrose isomerase was carried out according to the manufacturer'sdata on a 50 ml culture scale. After harvesting the cells the pellet wasresuspended in 1 ml 50 mM sodium phosphate buffer (pH 6.0) and thesoluble protein fraction was released by ultrasonication. An aliquot ofthe raw extract was mixed with the same volume of 600 mM sucrose andincubated for 24 hours at 30° C. An aliquot of the mixture was subjectedto a HPLC analysis to detect the palatinose produced. The chromatogramconfirmed the production of palatinose by detecting the recombinantsucrose isomerase in E. coli.

Example 11

[0202] In vivo Detection of the Sucrose Isomerase Activity in TransgenicPlants

[0203] The in vivo functionality of the sucrose isomerase in transgenicplants was detected as follows: ethanol extracts were produced from 0.5cm² leaf disks of untransformed tobacco plants and the transformants35S-cwIso (from Example 5) and were analysed by HPLC, and the sugarswere identified using the corresponding standards. As the chromatogramsshowed, the expression of the sucrose isomerase in the cell wallresulted in a substantial accumulation of palatinose in the analysedp35S-cwIso plants. The wild-type contains no palatinose, as also couldbe seen clearly from the chromatograms.

Example 12

[0204] Functional Detection and Biochemical Characterisation of thePalatinase Activity in E. coli

[0205] The functional characterisation of the palatinase gene wasimplemented by expression of the recombinant protein in E. coli. Forthis purpose the plasmid pQE-palQ was transformed in E. coli (XL-I blue,Stratagene). The expression of the recombinant protein was carried outaccording to the manufacturer's data (Qiagen, Hilden, Germany) on a 50ml culture scale. After harvesting the cells by centrifugation thepellet was resuspended in 1 ml 30 mM HEPES (pH 7.5) and the solubleprotein fraction was released by ultrasonication. 20 μl of the rawextract were mixed with 80 μl of 100 mM palatinose and incubated for 40minutes at 30° C. In order to detect the palatinase activity thereleased glucose was determined in an aliquot of the mixture by acoupled optical enzymatic test. Thus, the palatinase activity of therecombinant enzyme could be clearly detected. In further experiments itwas demonstrated that the enzyme evolves its highest activity at areaction temperature of 30° C. and a pH of 7.0. When the reaction ratewas analysed depending on the concentration of the substrate, a K_(m)value of 10 mM for palatinose and a maximum reaction rate at a substrateconcentration of 90 mM palatinose could be determined.

Example 13

[0206] PCR-Amplification of a Trehalulase from Erwinia rhapontici

[0207] The entire open reading frame of trehalulase was cloned by meansof polymerase chain reaction (Polymerase Chain Reaction, PCR). Thetemplate material was genomic DNA from E. rhapontici, which was isolatedaccording to a standard protocol. The amplification was carried outusing the specific primers: FB184 5′-GGGATCCGTGCAAACTGGTGGAAAGAG-3′ (SEQID NO:19) FB185 5′-GTCGACTTACCGCTGATAAATTTGTGC-3′ (SEQ ID NO:20)

[0208] The primers FB184 and FB185 comprise the bases 4-23 and1638-1659, respectively, of the coding region of the trehalulase gene.

[0209] For cloning the DNA into expression vectors the primersadditionally contain the following restriction sites: primer FB96 or FB184: BamHI; primer FB 185: SalI. The PCR reaction mix (100 μl) containedbacterial chromosomal DNA (1 μg), primers FB184 and FB185 (250 ng ineach case), Pfu DNA polymerase reaction buffer (10 μL, Stratagene), 200μM dNTPs (dATP, dCTP, dGTP, dTTP) and 2.5 units Pfu DNA polymerase(Stratagene). Prior to the initiation of the amplification cycles themixture was heated for 5 minutes to 95° C. The polymerisation steps (30cycles) were carried out in an automated T3-Thermocycler (Biometra)according to the following program: denaturation 95° C. (1 minute),annealing of the primers at 55° C. (40 seconds), polymerase reaction at72° C. (2 minutes). The amplicon was digested with BamHI and SalI andthe fragment was cloned into the vector pCR-Blunt (Invitrogen), whichresulted in the plasmid pCR-PalZ (see FIG. 6). The identity of theamplified DNA was verified by means of sequence analysis.

[0210] Fragment A contains the sequence of a trehalulase from E.rhapontici, which extends from nucleotide 4-1659 of the trehalulasegene.

Example 14

[0211] Production of the Plasmid pTA29-cwPalZ

[0212] In a procedure similar to that described in Example 7, the codingregion of the trehalulase gene from Erwinia rhapontici was fused to aleader peptide of a plant gene necessary for the transport into theendoplasmic reticulum (proteinase inhibitor II gene from potato (Solanumtuberosum, Keil et al. (1986) vide supra) under the control of theanther-specific promoter of the TA29 gene from tobacco. The so obtainedconstruct pTA29-cwPalZ consists of three fragments A, B and C (see FIG.3) and allows the expression of the trehalulase in the cell wall oftapetum cells.

[0213] Fragment A contains the TA29 promoter from Nicotiana tabacum. Thefragment contains the nucleotides −1477 to +57 relative to theinitiation of transcription of the TA29 gene (Seurinck et al. (1990)Nucleic Acids Res. 18:3403). It was amplified by means of PCR fromgenomic DNA of Nicotiana tabacum Var. Samsun NN. The amplification wascarried out using the specific primers: FB1585′-GAATTCGTTTGACAGCTTATCATCGAT-3′ (SEQ ID NO:17) and FB1595′-GGTACCAGCTAATTTCTTTAAGTAAA-3′. (SEQ ID NO:18)

[0214] For cloning the DNA into the expression cassette the primers alsohave the following restriction sites: primer FB 158, EcoRI; primer FB159, Asp718. The PCR reaction mix (100 μl) contained genomic DNA oftobacco (2 μg), primers FB158 and FB159 (250 ng in each case), Pfu DNApolymerase reaction buffer (10 μl, Stratagene), 200 μM dNTPs (dATP,dCTP, dGTP, dTTP) and 2.5 units of Pfu DNA polymerase (Stratagene).Prior to the initiation of the amplification cycles the mixture washeated for 5 min to 95° C. The polymerisation steps (35 cycles) werecarried out in an automated T3-Thermocycler (Biometra) according to thefollowing program: denaturation at 95° C. (1 minute), annealing of theprimers at 55° C. (40 seconds), polymerase reaction at 72° C. (2minutes). The amplicon was digested with the restriction enzymes EcoRIand Asp718 and ligated into the corresponding sites of the polylinker ofpUC18. The identity of the amplified DNA was verified by means ofsequence analysis.

[0215] Fragment B contains the nucleotides 923-1059 of a proteinaseinhibitor II gene from potato (Solanum tuberosum, Keil et al. (1986),vide supra), which is fused via a linker with the sequence ACC GAA TTGGG to the trehalulase gene from Erwinia rhapontici, which comprises thenucleotides 4-1659. By this means a leader peptide of a plant proteinrequired for the transport of proteins into the endoplasmic reticulum isN-terminally fused to the trehalulase sequence.

[0216] Fragment C contains the polyadenylation signal of gene 3 of theT-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3:835),nucleotides 11749-11939, which was isolated as a PvuII/HindIII fragmentfrom the plasmid pAGV 40 (Herrera-Estrella et al. (1983) Nature 303,209) and has been cloned after addition of SphI linkers to the PvuIIsite between the SphI- and HindIII-sites of the polylinker of pUC18.

[0217] The chimeric gene was then cloned as a EcoRI/HindIII fragmentbetween the EcoRI/HindIII sites of the plasmid pBIN19 (Bevan (1984)Nucleic Acids Res. 12, 8711).

[0218] In pTA29-cwPalZ (TA29=promoter of the TA29 gene from tobacco,cw=cell wall, PalZ−trehalulase) the coding region of the trehalulasegene from E. rhapontici is under anther-specific control, the geneproduct is transported into the ER.

[0219] Transgenic plants, which were transformed with pTA29-cwPalZ bymeans of agrobacterium-mediated gene transfer, showed no difference intheir phenotype compared to the wild-type. The daughter plants obtainedfrom crossing these plants with the male sterile plants from Example 6again showed the male fertile phenotype of the pTA29-cwPalZ parentplants.

Example 15

[0220] Site-Directed Mutagenesis of the Palatinase from E. rhapontici toOptimise Palatinase Expression in Transgenic Plants

[0221] In order to avoid glycosylation of the palatinase from Erhapontici in transgenic plants, suitable amino acids were substitutedby site-directed mutagenesis in the region of potential glycosylationsites. The plasmid pQE-palQ was used as template. As far as thepalatinase sequence is concerned, pQE-palQ corresponds to pCR-palQ, butis suitable for the expression of the palatinase sequence in E. coli.The reaction mixture (50 μl) for PCR-supported mutagenesis was composedas follows: 50 ng pQE-palQ DNA, 250 ng each of 5′ or 3′ primer, Pfu DNApolymerase reaction buffer (5 μl, Stratagene), 200 μM dNTPs (dATP, dCTP,dGTP, dTTP) and 2.5 units of Pfu-DNA-polymerase (Stratagene). Thepolymerisation steps (15 cycles) were carried out in an automatedT3-Thermocycler (Biometra) according to the following program:denaturation at 95° C. (30 seconds), annealing of the primers at 55° C.(1 minute), polymerase reaction at 72° C. (15 minutes). After completionof the reaction the parental DNA was digested with 1 unit of restrictionenzyme DpnI for 1 hour at 37° C. Then 1 μl of the mixture was used forthe transformation of E. coli.

[0222] For mutation 1 threonine at position 105 was substituted byalanine, thereby resulting in plasmid pQE-palQ T105A. 5′-Primer: SL365′-CTG GTG GTC AAC CAT GCC TCT GAC GAA CAT CCC-3′ (SEQ ID NO:21)3′-Primer: SL37 5′-GGG ATG TTC GTC AGA GGC ATG GTT GAC CAC CAG-3′ (SEQID NO:22)

[0223] For mutation 2 threonine at position 248 was substituted byalanine, thus resulting in plasmid pQE-palQ T248A. 5′-Primer: SL395′-GAG ACG TGG AGC GCA GCG CCA GAA GAC GCC CTG-3′ (SEQ ID NO:23)3′-Primer: SL40 5′-CAG GGC GTC TTC TGG CGC TGC GCT CCA CGT CTC-3′ (SEQID NO:24)

[0224] For mutation 3 threonine at position 502 was substituted byalanine, resulting in plasmid pQE-palQ T502A. 5′-Primer: SL42 5′-G GTGATC AAT AAC TTC GCG CGA GAC GCT GTG ATG C-3′ (SEQ ID NO:25) 3′-Primer:SL43 5′-G CAT CAC AGC GTC TCG CGC GAA GTT ATT GAT CAC C-3′ (SEQ IDNO:26)

[0225] The mutation event was in each case verified by sequencing thecorresponding region of the palatinase sequence. Functional expressionof the mutagenised enzyme in E. coli could demonstrate in all cases thatthe respective amino acid substitution does not have any disadvantageouseffect on the enzymatic activity. The mutations were then linked to eachother by the above-mentioned strategy so that a palatinase was finallyproduced which has no putative glycosylation sites left. Afterexpression in E. coli also this enzyme showed no disadvantageouscatalytic properties. (a) palQ wild-type100                 105                 110 amino acid sequence Leu ValVal Asn His Thr Ser Asp Glu His Pro (SEQ ID NO:27) nucleotide sequenceCTG GTG GTC AAC CAT ACC TCT GAC GAA CAT CCC (SEQ ID NO:28) (b) palQT105A amino acid sequence  •   •   •   •   •  Ala  •   •   •   •   •(SEQ ID NO:29) nucleotide sequence ... ... ... ... ... GCC ... ... ...... ... (SEQ ID NO:30) (c) palQ wild-type243                 248                 253 amino acid sequence Glu ThrTrp Ser Ala Thr Pro Glu Asp Ala Leu (SEQ ID NO:31) nucleotide sequenceGAG ACG TGG AGC GCA ACG CCA GAA GAC GCC CTG (SEQ ID NO:32) (d) palQT248A amino acid sequence  •   •   •   •   •  Ala  •   •   •   •   •(SEQ ID NO:33) nucleotide sequence ... ... ... ... ... GCC ... ... ...... ... (SEQ ID NO:34) (e) palQ wild-type497                 502                 507 amino acid sequence Val IleAsn Asn Phe Thr Arg Asp Ala Val Met (SEQ ID NO:35) nucleotide sequenceGTG ATC AAT AAC TTC ACG CGA GAC GCT GTG ATG (SEQ ID NO:36) (f) palQT502A amino acid sequence  •   •   •   •   •  Ala  •   •   •   •   •(SEQ ID NO:37) nucleotide sequence ... ... ... ... ... GCC ... ... ...... ... (SEQ ID NO:38)

[0226] The mutated palatinase sequence was subsequently subcloned into aplant transformation vector and was expressed in plants.

1 38 1 1656 DNA Erwinia rhapontici 1 atgcgcagca caccgcactg gaaagaggccgtggtttatc aggtctatcc gcgcagcttt 60 atggacagta acggcgacgg taccggcgatctcaacggta ttatcagcaa gctcgattac 120 ctgcaacagc tcggcatcac gctgttgtggctgtcgcccg tataccgttc gccgatggac 180 gataacggct atgacatctc tgattacgaagagattgccg acatttttgg ttcgatgagc 240 gacatggagc gcctgattgc agaagctaaagcgcgtgata tcgggatcct gatggatctg 300 gtggtcaacc atacctctga cgaacatccctggtttatcg acgcactgag ctcaaaaaac 360 agtgcttacc gtgactttta tatctggcgagcaccggcgg cagacggcgg gccgcctgat 420 gactctcgtt cgaactttgg tggcagtgcctggacgcttg atgaggccag cggtgaatac 480 tacctgcatc agttttccac gcgccagcccgatctcaact gggaaaaccc gcgcgttcgt 540 gaagccatcc acgccatgat gaaccgctggctggataagg gcatcggggg attccgaatg 600 gacgttatcg acctgatcgg gaaagaagtggatccacaga tcatggcgaa tggtcgtcat 660 cctcacctgt atcttcagca gatgaaccgggcgacctttg gcccgcgcgg cagcgtgacg 720 gtaggggaga cgtggagcgc aacgccagaagacgccctgc tctacagtgc cgaagagcgg 780 caagagcggc aagagctgac gatggtctttcagtttgagc acatcaaact tttctgggat 840 gaacagtacg ggaagtggtg taaccagccgtttgatctgt tgcgctttaa ggccgtgatt 900 gacaagtggc agacggcact ggctgaccatggctggaact cgttgttctg gagcaaccat 960 gatttgcctc gcgcggtctc caaatttggtgacgacggtg agtatcgcgt ggtatcagca 1020 aaaatgctcg ccaccgcgct tcactgccttaaaggcacac cttacattta tcagggtgaa 1080 gagattggca tgaccaacgt gaattttgctgatattgacg actatcggga tattgaaagc 1140 ctgaatcttt atcaggagcg gatcgccgaagggatgagcc acgaagcgat gatgcgcggt 1200 atccacgcca acgggcccga taatgcgcgaacgccaatgc agtggacagc agtccacatg 1260 ccgggtttac caccggtcag ccctggattgaggctaatcc taacttcagg acagtggaat 1320 gtcgcggctg cgcttgacga tcctgactctgttttttacc actaccagaa gctggtggca 1380 ttgcgtaagc agctgccgct gctggtgcacggcgacttca ggcagatcgt tgtcgaacat 1440 ccgcaggtgt ttgcctggct gcgcacgctgggggagcaga cgctggtggt gatcaataac 1500 ttcacgcgag acgctgtgat gctggcgatccccgacaatc tgcagagcca gcagggccgt 1560 tgtctcatca acaattacgc gccacgggagcagttggagc cgattatgga actgcaacct 1620 tatgaatcct ttgcattact tattgagaggctgtga 1656 2 1656 DNA Erwinia rhapontici CDS (1)..(1653) 2 atg cgc agcaca ccg cac tgg aaa gag gcc gtg gtt tat cag gtc tat 48 Met Arg Ser ThrPro His Trp Lys Glu Ala Val Val Tyr Gln Val Tyr 1 5 10 15 ccg cgc agcttt atg gac agt aac ggc gac ggt acc ggc gat ctc aac 96 Pro Arg Ser PheMet Asp Ser Asn Gly Asp Gly Thr Gly Asp Leu Asn 20 25 30 ggt att atc agcaag ctc gat tac ctg caa cag ctc ggc atc acg ctg 144 Gly Ile Ile Ser LysLeu Asp Tyr Leu Gln Gln Leu Gly Ile Thr Leu 35 40 45 ttg tgg ctg tcg cccgta tac cgt tcg ccg atg gac gat aac ggc tat 192 Leu Trp Leu Ser Pro ValTyr Arg Ser Pro Met Asp Asp Asn Gly Tyr 50 55 60 gac atc tct gat tac gaagag att gcc gac att ttt ggt tcg atg agc 240 Asp Ile Ser Asp Tyr Glu GluIle Ala Asp Ile Phe Gly Ser Met Ser 65 70 75 80 gac atg gag cgc ctg attgca gaa gct aaa gcg cgt gat atc ggg atc 288 Asp Met Glu Arg Leu Ile AlaGlu Ala Lys Ala Arg Asp Ile Gly Ile 85 90 95 ctg atg gat ctg gtg gtc aaccat acc tct gac gaa cat ccc tgg ttt 336 Leu Met Asp Leu Val Val Asn HisThr Ser Asp Glu His Pro Trp Phe 100 105 110 atc gac gca ctg agc tca aaaaac agt gct tac cgt gac ttt tat atc 384 Ile Asp Ala Leu Ser Ser Lys AsnSer Ala Tyr Arg Asp Phe Tyr Ile 115 120 125 tgg cga gca ccg gcg gca gacggc ggg ccg cct gat gac tct cgt tcg 432 Trp Arg Ala Pro Ala Ala Asp GlyGly Pro Pro Asp Asp Ser Arg Ser 130 135 140 aac ttt ggt ggc agt gcc tggacg ctt gat gag gcc agc ggt gaa tac 480 Asn Phe Gly Gly Ser Ala Trp ThrLeu Asp Glu Ala Ser Gly Glu Tyr 145 150 155 160 tac ctg cat cag ttt tccacg cgc cag ccc gat ctc aac tgg gaa aac 528 Tyr Leu His Gln Phe Ser ThrArg Gln Pro Asp Leu Asn Trp Glu Asn 165 170 175 ccg cgc gtt cgt gaa gccatc cac gcc atg atg aac cgc tgg ctg gat 576 Pro Arg Val Arg Glu Ala IleHis Ala Met Met Asn Arg Trp Leu Asp 180 185 190 aag ggc atc ggg gga ttccga atg gac gtt atc gac ctg atc ggg aaa 624 Lys Gly Ile Gly Gly Phe ArgMet Asp Val Ile Asp Leu Ile Gly Lys 195 200 205 gaa gtg gat cca cag atcatg gcg aat ggt cgt cat cct cac ctg tat 672 Glu Val Asp Pro Gln Ile MetAla Asn Gly Arg His Pro His Leu Tyr 210 215 220 ctt cag cag atg aac cgggcg acc ttt ggc ccg cgc ggc agc gtg acg 720 Leu Gln Gln Met Asn Arg AlaThr Phe Gly Pro Arg Gly Ser Val Thr 225 230 235 240 gta ggg gag acg tggagc gca acg cca gaa gac gcc ctg ctc tac agt 768 Val Gly Glu Thr Trp SerAla Thr Pro Glu Asp Ala Leu Leu Tyr Ser 245 250 255 gcc gaa gag cgg caagag cgg caa gag ctg acg atg gtc ttt cag ttt 816 Ala Glu Glu Arg Gln GluArg Gln Glu Leu Thr Met Val Phe Gln Phe 260 265 270 gag cac atc aaa cttttc tgg gat gaa cag tac ggg aag tgg tgt aac 864 Glu His Ile Lys Leu PheTrp Asp Glu Gln Tyr Gly Lys Trp Cys Asn 275 280 285 cag ccg ttt gat ctgttg cgc ttt aag gcc gtg att gac aag tgg cag 912 Gln Pro Phe Asp Leu LeuArg Phe Lys Ala Val Ile Asp Lys Trp Gln 290 295 300 acg gca ctg gct gaccat ggc tgg aac tcg ttg ttc tgg agc aac cat 960 Thr Ala Leu Ala Asp HisGly Trp Asn Ser Leu Phe Trp Ser Asn His 305 310 315 320 gat ttg cct cgcgcg gtc tcc aaa ttt ggt gac gac ggt gag tat cgc 1008 Asp Leu Pro Arg AlaVal Ser Lys Phe Gly Asp Asp Gly Glu Tyr Arg 325 330 335 gtg gta tca gcaaaa atg ctc gcc acc gcg ctt cac tgc ctt aaa ggc 1056 Val Val Ser Ala LysMet Leu Ala Thr Ala Leu His Cys Leu Lys Gly 340 345 350 aca cct tac atttat cag ggt gaa gag att ggc atg acc aac gtg aat 1104 Thr Pro Tyr Ile TyrGln Gly Glu Glu Ile Gly Met Thr Asn Val Asn 355 360 365 ttt gct gat attgac gac tat cgg gat att gaa agc ctg aat ctt tat 1152 Phe Ala Asp Ile AspAsp Tyr Arg Asp Ile Glu Ser Leu Asn Leu Tyr 370 375 380 cag gag cgg atcgcc gaa ggg atg agc cac gaa gcg atg atg cgc ggt 1200 Gln Glu Arg Ile AlaGlu Gly Met Ser His Glu Ala Met Met Arg Gly 385 390 395 400 atc cac gccaac ggg ccc gat aat gcg cga acg cca atg cag tgg aca 1248 Ile His Ala AsnGly Pro Asp Asn Ala Arg Thr Pro Met Gln Trp Thr 405 410 415 gca gtc cacatg ccg ggt tta cca ccg gtc agc cct gga ttg agg cta 1296 Ala Val His MetPro Gly Leu Pro Pro Val Ser Pro Gly Leu Arg Leu 420 425 430 atc cta acttca gga cag tgg aat gtc gcg gct gcg ctt gac gat cct 1344 Ile Leu Thr SerGly Gln Trp Asn Val Ala Ala Ala Leu Asp Asp Pro 435 440 445 gac tct gttttt tac cac tac cag aag ctg gtg gca ttg cgt aag cag 1392 Asp Ser Val PheTyr His Tyr Gln Lys Leu Val Ala Leu Arg Lys Gln 450 455 460 ctg ccg ctgctg gtg cac ggc gac ttc agg cag atc gtt gtc gaa cat 1440 Leu Pro Leu LeuVal His Gly Asp Phe Arg Gln Ile Val Val Glu His 465 470 475 480 ccg caggtg ttt gcc tgg ctg cgc acg ctg ggg gag cag acg ctg gtg 1488 Pro Gln ValPhe Ala Trp Leu Arg Thr Leu Gly Glu Gln Thr Leu Val 485 490 495 gtg atcaat aac ttc acg cga gac gct gtg atg ctg gcg atc ccc gac 1536 Val Ile AsnAsn Phe Thr Arg Asp Ala Val Met Leu Ala Ile Pro Asp 500 505 510 aat ctgcag agc cag cag ggc cgt tgt ctc atc aac aat tac gcg cca 1584 Asn Leu GlnSer Gln Gln Gly Arg Cys Leu Ile Asn Asn Tyr Ala Pro 515 520 525 cgg gagcag ttg gag ccg att atg gaa ctg caa cct tat gaa tcc ttt 1632 Arg Glu GlnLeu Glu Pro Ile Met Glu Leu Gln Pro Tyr Glu Ser Phe 530 535 540 gca ttactt att gag agg ctg tga 1656 Ala Leu Leu Ile Glu Arg Leu 545 550 3 551PRT Erwinia rhapontici 3 Met Arg Ser Thr Pro His Trp Lys Glu Ala Val ValTyr Gln Val Tyr 1 5 10 15 Pro Arg Ser Phe Met Asp Ser Asn Gly Asp GlyThr Gly Asp Leu Asn 20 25 30 Gly Ile Ile Ser Lys Leu Asp Tyr Leu Gln GlnLeu Gly Ile Thr Leu 35 40 45 Leu Trp Leu Ser Pro Val Tyr Arg Ser Pro MetAsp Asp Asn Gly Tyr 50 55 60 Asp Ile Ser Asp Tyr Glu Glu Ile Ala Asp IlePhe Gly Ser Met Ser 65 70 75 80 Asp Met Glu Arg Leu Ile Ala Glu Ala LysAla Arg Asp Ile Gly Ile 85 90 95 Leu Met Asp Leu Val Val Asn His Thr SerAsp Glu His Pro Trp Phe 100 105 110 Ile Asp Ala Leu Ser Ser Lys Asn SerAla Tyr Arg Asp Phe Tyr Ile 115 120 125 Trp Arg Ala Pro Ala Ala Asp GlyGly Pro Pro Asp Asp Ser Arg Ser 130 135 140 Asn Phe Gly Gly Ser Ala TrpThr Leu Asp Glu Ala Ser Gly Glu Tyr 145 150 155 160 Tyr Leu His Gln PheSer Thr Arg Gln Pro Asp Leu Asn Trp Glu Asn 165 170 175 Pro Arg Val ArgGlu Ala Ile His Ala Met Met Asn Arg Trp Leu Asp 180 185 190 Lys Gly IleGly Gly Phe Arg Met Asp Val Ile Asp Leu Ile Gly Lys 195 200 205 Glu ValAsp Pro Gln Ile Met Ala Asn Gly Arg His Pro His Leu Tyr 210 215 220 LeuGln Gln Met Asn Arg Ala Thr Phe Gly Pro Arg Gly Ser Val Thr 225 230 235240 Val Gly Glu Thr Trp Ser Ala Thr Pro Glu Asp Ala Leu Leu Tyr Ser 245250 255 Ala Glu Glu Arg Gln Glu Arg Gln Glu Leu Thr Met Val Phe Gln Phe260 265 270 Glu His Ile Lys Leu Phe Trp Asp Glu Gln Tyr Gly Lys Trp CysAsn 275 280 285 Gln Pro Phe Asp Leu Leu Arg Phe Lys Ala Val Ile Asp LysTrp Gln 290 295 300 Thr Ala Leu Ala Asp His Gly Trp Asn Ser Leu Phe TrpSer Asn His 305 310 315 320 Asp Leu Pro Arg Ala Val Ser Lys Phe Gly AspAsp Gly Glu Tyr Arg 325 330 335 Val Val Ser Ala Lys Met Leu Ala Thr AlaLeu His Cys Leu Lys Gly 340 345 350 Thr Pro Tyr Ile Tyr Gln Gly Glu GluIle Gly Met Thr Asn Val Asn 355 360 365 Phe Ala Asp Ile Asp Asp Tyr ArgAsp Ile Glu Ser Leu Asn Leu Tyr 370 375 380 Gln Glu Arg Ile Ala Glu GlyMet Ser His Glu Ala Met Met Arg Gly 385 390 395 400 Ile His Ala Asn GlyPro Asp Asn Ala Arg Thr Pro Met Gln Trp Thr 405 410 415 Ala Val His MetPro Gly Leu Pro Pro Val Ser Pro Gly Leu Arg Leu 420 425 430 Ile Leu ThrSer Gly Gln Trp Asn Val Ala Ala Ala Leu Asp Asp Pro 435 440 445 Asp SerVal Phe Tyr His Tyr Gln Lys Leu Val Ala Leu Arg Lys Gln 450 455 460 LeuPro Leu Leu Val His Gly Asp Phe Arg Gln Ile Val Val Glu His 465 470 475480 Pro Gln Val Phe Ala Trp Leu Arg Thr Leu Gly Glu Gln Thr Leu Val 485490 495 Val Ile Asn Asn Phe Thr Arg Asp Ala Val Met Leu Ala Ile Pro Asp500 505 510 Asn Leu Gln Ser Gln Gln Gly Arg Cys Leu Ile Asn Asn Tyr AlaPro 515 520 525 Arg Glu Gln Leu Glu Pro Ile Met Glu Leu Gln Pro Tyr GluSer Phe 530 535 540 Ala Leu Leu Ile Glu Arg Leu 545 550 4 1803 DNAErwinia rhapontici 4 atgtcctctc aaggattgaa aacggctgtc gctatttttcttgcaaccac tttttctgcc 60 acatcctatc aggcctgcag tgccgggcca gataccgccccctcactcac cgttcagcaa 120 tcaaatgccc tgcccacatg gtggaagcag gctgttttttatcaggtata tccacgctca 180 tttaaagata cgaatgggga tggcattggg gatttaaacggtattattga gaatttagac 240 tatctgaaga aactgggtat tgatgcgatt tggatcaatccacattacga ttcgccgaat 300 acggataatg gttatgacat ccgggattac cgtaagataatgaaagaata cggtacgatg 360 gaagactttg accgtcttat ttcagaaatg aagaaacgcaatatgcgttt gatgattgat 420 attgttatca accacaccag cgatcagcat gcctggtttgttcagagcaa atcgggtaag 480 aacaacccct acagggacta ttacttctgg cgtgacggtaaggatggcca tgcccccaat 540 aactatccct ccttcttcgg tggctcagcc tgggaaaaagacgataaatc aggccagtat 600 tacctccatt actttgccaa acagcaaccc gacctcaactgggacaatcc caaagtccgt 660 caagacctgt atgacatgct ccgcttctgg ttagataaaggcgtttctgg tttacgcttt 720 gataccgttg ccacctactc gaaaatcccg aacttccctgaccttagcca acagcagtta 780 aaaaatttcg ccgaggaata tactaaaggt cctaaaattcacgactacgt gaatgaaatg 840 aacagagaag tattatccca ctatgatatc gccactgcgggggaaatatt tggggttcct 900 ctggataaat cgattaagtt tttcgatcgc cgtagaaatgaattaaatat agcgtttacg 960 tttgatctga tcaggctcga tcgtgatgct gatgaaagatggcggcgaaa agactggacc 1020 ctttcgcagt tccgaaaaat tgtcgataag gttgaccaaacggcaggaga gtatgggtgg 1080 aatgcctttt tcttagacaa tcacgacaat ccccgcgcggtttctcactt tggtgatgat 1140 cgaccacaat ggcgcgagca tgcggcgaaa gcactggcaacattgacgct gacccagcgt 1200 gcaacgccgt ttatctatca gggttcagaa ctcggtatgaccaattatcc ctttaaaaaa 1260 atcgatgatt tcgatgatgt agaggtgaaa ggtttttggcaagactacgt tgaaacaggc 1320 aaagtgaaag ctgaggaatt ccttcaaaac gtacgccaaaccagccgtga taacagcaga 1380 acccccttcc agtgggatgc aagcaaaaac gcgggctttaccagtggaac cccctggtta 1440 aaaatcaatc ccaattataa agaaatcaac agcgcagatcagattaataa tccaaattcc 1500 gtatttaact attatagaaa gctgattaac attcgccatgacatccctgc cttgacctac 1560 ggcagttata ttgatttaga ccctgacaac aattcagtctatgcttacac ccgaacgctc 1620 ggcgctgaaa aatatcttgt ggtcattaat tttaaagaagaagtgatgca ctacaccctg 1680 cccggggatt tatccatcaa taaggtgatt actgaaaacaacagtcacac tattgtgaat 1740 aaaaatgaca ggcaactccg tcttgaaccc tggcagtcgggcatttataa acttaatccg 1800 tag 1803 5 1803 DNA Erwinia rhapontici CDS(1)..(1800) 5 atg tcc tct caa gga ttg aaa acg gct gtc gct att ttt cttgca acc 48 Met Ser Ser Gln Gly Leu Lys Thr Ala Val Ala Ile Phe Leu AlaThr 1 5 10 15 act ttt tct gcc aca tcc tat cag gcc tgc agt gcc ggg ccagat acc 96 Thr Phe Ser Ala Thr Ser Tyr Gln Ala Cys Ser Ala Gly Pro AspThr 20 25 30 gcc ccc tca ctc acc gtt cag caa tca aat gcc ctg ccc aca tggtgg 144 Ala Pro Ser Leu Thr Val Gln Gln Ser Asn Ala Leu Pro Thr Trp Trp35 40 45 aag cag gct gtt ttt tat cag gta tat cca cgc tca ttt aaa gat acg192 Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr 5055 60 aat ggg gat ggc att ggg gat tta aac ggt att att gag aat tta gac240 Asn Gly Asp Gly Ile Gly Asp Leu Asn Gly Ile Ile Glu Asn Leu Asp 6570 75 80 tat ctg aag aaa ctg ggt att gat gcg att tgg atc aat cca cat tac288 Tyr Leu Lys Lys Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr 8590 95 gat tcg ccg aat acg gat aat ggt tat gac atc cgg gat tac cgt aag336 Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys 100105 110 ata atg aaa gaa tac ggt acg atg gaa gac ttt gac cgt ctt att tca384 Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser 115120 125 gaa atg aag aaa cgc aat atg cgt ttg atg att gat att gtt atc aac432 Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Ile Val Ile Asn 130135 140 cac acc agc gat cag cat gcc tgg ttt gtt cag agc aaa tcg ggt aag480 His Thr Ser Asp Gln His Ala Trp Phe Val Gln Ser Lys Ser Gly Lys 145150 155 160 aac aac ccc tac agg gac tat tac ttc tgg cgt gac ggt aag gatggc 528 Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Gly165 170 175 cat gcc ccc aat aac tat ccc tcc ttc ttc ggt ggc tca gcc tgggaa 576 His Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glu180 185 190 aaa gac gat aaa tca ggc cag tat tac ctc cat tac ttt gcc aaacag 624 Lys Asp Asp Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln195 200 205 caa ccc gac ctc aac tgg gac aat ccc aaa gtc cgt caa gac ctgtat 672 Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr210 215 220 gac atg ctc cgc ttc tgg tta gat aaa ggc gtt tct ggt tta cgcttt 720 Asp Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe225 230 235 240 gat acc gtt gcc acc tac tcg aaa atc ccg aac ttc cct gacctt agc 768 Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asn Phe Pro Asp LeuSer 245 250 255 caa cag cag tta aaa aat ttc gcc gag gaa tat act aaa ggtcct aaa 816 Gln Gln Gln Leu Lys Asn Phe Ala Glu Glu Tyr Thr Lys Gly ProLys 260 265 270 att cac gac tac gtg aat gaa atg aac aga gaa gta tta tcccac tat 864 Ile His Asp Tyr Val Asn Glu Met Asn Arg Glu Val Leu Ser HisTyr 275 280 285 gat atc gcc act gcg ggg gaa ata ttt ggg gtt cct ctg gataaa tcg 912 Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp LysSer 290 295 300 att aag ttt ttc gat cgc cgt aga aat gaa tta aat ata gcgttt acg 960 Ile Lys Phe Phe Asp Arg Arg Arg Asn Glu Leu Asn Ile Ala PheThr 305 310 315 320 ttt gat ctg atc agg ctc gat cgt gat gct gat gaa agatgg cgg cga 1008 Phe Asp Leu Ile Arg Leu Asp Arg Asp Ala Asp Glu Arg TrpArg Arg 325 330 335 aaa gac tgg acc ctt tcg cag ttc cga aaa att gtc gataag gtt gac 1056 Lys Asp Trp Thr Leu Ser Gln Phe Arg Lys Ile Val Asp LysVal Asp 340 345 350 caa acg gca gga gag tat ggg tgg aat gcc ttt ttc ttagac aat cac 1104 Gln Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu AspAsn His 355 360 365 gac aat ccc cgc gcg gtt tct cac ttt ggt gat gat cgacca caa tgg 1152 Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg ProGln Trp 370 375 380 cgc gag cat gcg gcg aaa gca ctg gca aca ttg acg ctgacc cag cgt 1200 Arg Glu His Ala Ala Lys Ala Leu Ala Thr Leu Thr Leu ThrGln Arg 385 390 395 400 gca acg ccg ttt atc tat cag ggt tca gaa ctc ggtatg acc aat tat 1248 Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly MetThr Asn Tyr 405 410 415 ccc ttt aaa aaa atc gat gat ttc gat gat gta gaggtg aaa ggt ttt 1296 Pro Phe Lys Lys Ile Asp Asp Phe Asp Asp Val Glu ValLys Gly Phe 420 425 430 tgg caa gac tac gtt gaa aca ggc aaa gtg aaa gctgag gaa ttc ctt 1344 Trp Gln Asp Tyr Val Glu Thr Gly Lys Val Lys Ala GluGlu Phe Leu 435 440 445 caa aac gta cgc caa acc agc cgt gat aac agc agaacc ccc ttc cag 1392 Gln Asn Val Arg Gln Thr Ser Arg Asp Asn Ser Arg ThrPro Phe Gln 450 455 460 tgg gat gca agc aaa aac gcg ggc ttt acc agt ggaacc ccc tgg tta 1440 Trp Asp Ala Ser Lys Asn Ala Gly Phe Thr Ser Gly ThrPro Trp Leu 465 470 475 480 aaa atc aat ccc aat tat aaa gaa atc aac agcgca gat cag att aat 1488 Lys Ile Asn Pro Asn Tyr Lys Glu Ile Asn Ser AlaAsp Gln Ile Asn 485 490 495 aat cca aat tcc gta ttt aac tat tat aga aagctg att aac att cgc 1536 Asn Pro Asn Ser Val Phe Asn Tyr Tyr Arg Lys LeuIle Asn Ile Arg 500 505 510 cat gac atc cct gcc ttg acc tac ggc agt tatatt gat tta gac cct 1584 His Asp Ile Pro Ala Leu Thr Tyr Gly Ser Tyr IleAsp Leu Asp Pro 515 520 525 gac aac aat tca gtc tat gct tac acc cga acgctc ggc gct gaa aaa 1632 Asp Asn Asn Ser Val Tyr Ala Tyr Thr Arg Thr LeuGly Ala Glu Lys 530 535 540 tat ctt gtg gtc att aat ttt aaa gaa gaa gtgatg cac tac acc ctg 1680 Tyr Leu Val Val Ile Asn Phe Lys Glu Glu Val MetHis Tyr Thr Leu 545 550 555 560 ccc ggg gat tta tcc atc aat aag gtg attact gaa aac aac agt cac 1728 Pro Gly Asp Leu Ser Ile Asn Lys Val Ile ThrGlu Asn Asn Ser His 565 570 575 act att gtg aat aaa aat gac agg caa ctccgt ctt gaa ccc tgg cag 1776 Thr Ile Val Asn Lys Asn Asp Arg Gln Leu ArgLeu Glu Pro Trp Gln 580 585 590 tcg ggc att tat aaa ctt aat ccg tag 1803Ser Gly Ile Tyr Lys Leu Asn Pro 595 600 6 600 PRT Erwinia rhapontici 6Met Ser Ser Gln Gly Leu Lys Thr Ala Val Ala Ile Phe Leu Ala Thr 1 5 1015 Thr Phe Ser Ala Thr Ser Tyr Gln Ala Cys Ser Ala Gly Pro Asp Thr 20 2530 Ala Pro Ser Leu Thr Val Gln Gln Ser Asn Ala Leu Pro Thr Trp Trp 35 4045 Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr 50 5560 Asn Gly Asp Gly Ile Gly Asp Leu Asn Gly Ile Ile Glu Asn Leu Asp 65 7075 80 Tyr Leu Lys Lys Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr 8590 95 Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys100 105 110 Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu IleSer 115 120 125 Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Ile ValIle Asn 130 135 140 His Thr Ser Asp Gln His Ala Trp Phe Val Gln Ser LysSer Gly Lys 145 150 155 160 Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp ArgAsp Gly Lys Asp Gly 165 170 175 His Ala Pro Asn Asn Tyr Pro Ser Phe PheGly Gly Ser Ala Trp Glu 180 185 190 Lys Asp Asp Lys Ser Gly Gln Tyr TyrLeu His Tyr Phe Ala Lys Gln 195 200 205 Gln Pro Asp Leu Asn Trp Asp AsnPro Lys Val Arg Gln Asp Leu Tyr 210 215 220 Asp Met Leu Arg Phe Trp LeuAsp Lys Gly Val Ser Gly Leu Arg Phe 225 230 235 240 Asp Thr Val Ala ThrTyr Ser Lys Ile Pro Asn Phe Pro Asp Leu Ser 245 250 255 Gln Gln Gln LeuLys Asn Phe Ala Glu Glu Tyr Thr Lys Gly Pro Lys 260 265 270 Ile His AspTyr Val Asn Glu Met Asn Arg Glu Val Leu Ser His Tyr 275 280 285 Asp IleAla Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Lys Ser 290 295 300 IleLys Phe Phe Asp Arg Arg Arg Asn Glu Leu Asn Ile Ala Phe Thr 305 310 315320 Phe Asp Leu Ile Arg Leu Asp Arg Asp Ala Asp Glu Arg Trp Arg Arg 325330 335 Lys Asp Trp Thr Leu Ser Gln Phe Arg Lys Ile Val Asp Lys Val Asp340 345 350 Gln Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp AsnHis 355 360 365 Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg ProGln Trp 370 375 380 Arg Glu His Ala Ala Lys Ala Leu Ala Thr Leu Thr LeuThr Gln Arg 385 390 395 400 Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu LeuGly Met Thr Asn Tyr 405 410 415 Pro Phe Lys Lys Ile Asp Asp Phe Asp AspVal Glu Val Lys Gly Phe 420 425 430 Trp Gln Asp Tyr Val Glu Thr Gly LysVal Lys Ala Glu Glu Phe Leu 435 440 445 Gln Asn Val Arg Gln Thr Ser ArgAsp Asn Ser Arg Thr Pro Phe Gln 450 455 460 Trp Asp Ala Ser Lys Asn AlaGly Phe Thr Ser Gly Thr Pro Trp Leu 465 470 475 480 Lys Ile Asn Pro AsnTyr Lys Glu Ile Asn Ser Ala Asp Gln Ile Asn 485 490 495 Asn Pro Asn SerVal Phe Asn Tyr Tyr Arg Lys Leu Ile Asn Ile Arg 500 505 510 His Asp IlePro Ala Leu Thr Tyr Gly Ser Tyr Ile Asp Leu Asp Pro 515 520 525 Asp AsnAsn Ser Val Tyr Ala Tyr Thr Arg Thr Leu Gly Ala Glu Lys 530 535 540 TyrLeu Val Val Ile Asn Phe Lys Glu Glu Val Met His Tyr Thr Leu 545 550 555560 Pro Gly Asp Leu Ser Ile Asn Lys Val Ile Thr Glu Asn Asn Ser His 565570 575 Thr Ile Val Asn Lys Asn Asp Arg Gln Leu Arg Leu Glu Pro Trp Gln580 585 590 Ser Gly Ile Tyr Lys Leu Asn Pro 595 600 7 1659 DNA Erwiniarhapontici 7 atggcaaact ggtggaaaga ggccgtggcg tatcagatat acccgcgcagcttcaacgac 60 agcaataacg atggcattgg tgacctgaat ggcatcacgg aaaaactcgattacctggaa 120 gatttgggca tcgatctgat ttggatctgc cccatgtatc agtcccccaatgatgacaac 180 ggctatgaca tcagcgatta ccagaaaatc atggctgagt ttggcacgatggacgatttt 240 gaccgtctgc ttgaacaggt gcatgcgcgc ggtatgcgcc tgattattgatttagtggtg 300 aaccatactt ctgatgagca tccgtggttt ttagcgtcca gcgcatcacgggataacccg 360 aaacgcgact ggtacatctg gcgcgacggt aaagcgggcg ctgagccgaacaactgggaa 420 agcatcttca acggttctgc ctggaaatac agcgcggcga ccgggcagtatttcctgcat 480 ttgttctccg aaaagcagcc agatttgaac tgggaaaacc ccgaggtgcgttcggcggtg 540 tatgccatga tgcgttggtg gcttgacaaa ggggtagatg gttttcgcattgatgccatc 600 tgccatatga aaaaagagcc gactttcagc gatatgccta atcccctggcgctgccttac 660 gtaccgtcat tcgagcgcca cctcaactac gacggcctgc ttgattacgtcgatgacatg 720 tgtgaacagg tgttcagtca ctatgacatt gtgaccatcg gcgaaatgaacggtgcctcc 780 gctgaacagg gtgaagagtg ggtcggcgag cagcggggca ggctgaatatgatctttcag 840 tttgagcacg tgaagctgtg gcaggatggg caaaaggaca ccctggaggccagtctcgat 900 ttacccggct taaaagagat tttcacgcgc tggcagacac tgctggaaaacaaaggctgg 960 aacgcgttat acgtagaaaa tcatgatctt ccccgtgtgg tatcgggctggggcgacgat 1020 aaaaattatc aacgtgaaag cgcgaccgcc attgcggcga tgttcttcctgatgaaaggt 1080 acgccgttta tttatcaggg gcaggaactt ggcatgacca atacgcatttcgccagcctg 1140 gaggattttg acgacgttgc cgcgaagaaa ctcgccgttg aaatgcgccgacagggcagg 1200 gaagagcccg agatcctcgc cttcctcagc cgcaccgggc gcgaaaactcgcgcaccccg 1260 atgcagtcgg atcagagtgc ccacggcggt ttcagcaatg ctaccccctggtttcctgcg 1320 aacagtaatt accctgtaat caacgtggcg gatcaacgtg ctgacagcggttccgtgctg 1380 aacttctatc gtgcgctcat tcgcctgcgc cggcagatgc cggtattgattgaaggggct 1440 tatcaacttc tgctgccgac acatccgcag atctatgcct atacccgtcgtctgaatgaa 1500 cagcaggtgt tggtgatcgt caatttcagt gcccatcagc aggagatagatccgcaacag 1560 ctgttactgg acggctggca accgctgctg agcaattatc aggagcaggggaaacggcaa 1620 atcttacggg cttatgaggc acaaatttat cagcggtaa 1659 8 1659DNA Erwinia rhapontici CDS (1)..(1656) 8 atg gca aac tgg tgg aaa gag gccgtg gcg tat cag ata tac ccg cgc 48 Met Ala Asn Trp Trp Lys Glu Ala ValAla Tyr Gln Ile Tyr Pro Arg 1 5 10 15 agc ttc aac gac agc aat aac gatggc att ggt gac ctg aat ggc atc 96 Ser Phe Asn Asp Ser Asn Asn Asp GlyIle Gly Asp Leu Asn Gly Ile 20 25 30 acg gaa aaa ctc gat tac ctg gaa gatttg ggc atc gat ctg att tgg 144 Thr Glu Lys Leu Asp Tyr Leu Glu Asp LeuGly Ile Asp Leu Ile Trp 35 40 45 atc tgc ccc atg tat cag tcc ccc aat gatgac aac ggc tat gac atc 192 Ile Cys Pro Met Tyr Gln Ser Pro Asn Asp AspAsn Gly Tyr Asp Ile 50 55 60 agc gat tac cag aaa atc atg gct gag ttt ggcacg atg gac gat ttt 240 Ser Asp Tyr Gln Lys Ile Met Ala Glu Phe Gly ThrMet Asp Asp Phe 65 70 75 80 gac cgt ctg ctt gaa cag gtg cat gcg cgc ggtatg cgc ctg att att 288 Asp Arg Leu Leu Glu Gln Val His Ala Arg Gly MetArg Leu Ile Ile 85 90 95 gat tta gtg gtg aac cat act tct gat gag cat ccgtgg ttt tta gcg 336 Asp Leu Val Val Asn His Thr Ser Asp Glu His Pro TrpPhe Leu Ala 100 105 110 tcc agc gca tca cgg gat aac ccg aaa cgc gac tggtac atc tgg cgc 384 Ser Ser Ala Ser Arg Asp Asn Pro Lys Arg Asp Trp TyrIle Trp Arg 115 120 125 gac ggt aaa gcg ggc gct gag ccg aac aac tgg gaaagc atc ttc aac 432 Asp Gly Lys Ala Gly Ala Glu Pro Asn Asn Trp Glu SerIle Phe Asn 130 135 140 ggt tct gcc tgg aaa tac agc gcg gcg acc ggg cagtat ttc ctg cat 480 Gly Ser Ala Trp Lys Tyr Ser Ala Ala Thr Gly Gln TyrPhe Leu His 145 150 155 160 ttg ttc tcc gaa aag cag cca gat ttg aac tgggaa aac ccc gag gtg 528 Leu Phe Ser Glu Lys Gln Pro Asp Leu Asn Trp GluAsn Pro Glu Val 165 170 175 cgt tcg gcg gtg tat gcc atg atg cgt tgg tggctt gac aaa ggg gta 576 Arg Ser Ala Val Tyr Ala Met Met Arg Trp Trp LeuAsp Lys Gly Val 180 185 190 gat ggt ttt cgc att gat gcc atc tgc cat atgaaa aaa gag ccg act 624 Asp Gly Phe Arg Ile Asp Ala Ile Cys His Met LysLys Glu Pro Thr 195 200 205 ttc agc gat atg cct aat ccc ctg gcg ctg ccttac gta ccg tca ttc 672 Phe Ser Asp Met Pro Asn Pro Leu Ala Leu Pro TyrVal Pro Ser Phe 210 215 220 gag cgc cac ctc aac tac gac ggc ctg ctt gattac gtc gat gac atg 720 Glu Arg His Leu Asn Tyr Asp Gly Leu Leu Asp TyrVal Asp Asp Met 225 230 235 240 tgt gaa cag gtg ttc agt cac tat gac attgtg acc atc ggc gaa atg 768 Cys Glu Gln Val Phe Ser His Tyr Asp Ile ValThr Ile Gly Glu Met 245 250 255 aac ggt gcc tcc gct gaa cag ggt gaa gagtgg gtc ggc gag cag cgg 816 Asn Gly Ala Ser Ala Glu Gln Gly Glu Glu TrpVal Gly Glu Gln Arg 260 265 270 ggc agg ctg aat atg atc ttt cag ttt gagcac gtg aag ctg tgg cag 864 Gly Arg Leu Asn Met Ile Phe Gln Phe Glu HisVal Lys Leu Trp Gln 275 280 285 gat ggg caa aag gac acc ctg gag gcc agtctc gat tta ccc ggc tta 912 Asp Gly Gln Lys Asp Thr Leu Glu Ala Ser LeuAsp Leu Pro Gly Leu 290 295 300 aaa gag att ttc acg cgc tgg cag aca ctgctg gaa aac aaa ggc tgg 960 Lys Glu Ile Phe Thr Arg Trp Gln Thr Leu LeuGlu Asn Lys Gly Trp 305 310 315 320 aac gcg tta tac gta gaa aat cat gatctt ccc cgt gtg gta tcg ggc 1008 Asn Ala Leu Tyr Val Glu Asn His Asp LeuPro Arg Val Val Ser Gly 325 330 335 tgg ggc gac gat aaa aat tat caa cgtgaa agc gcg acc gcc att gcg 1056 Trp Gly Asp Asp Lys Asn Tyr Gln Arg GluSer Ala Thr Ala Ile Ala 340 345 350 gcg atg ttc ttc ctg atg aaa ggt acgccg ttt att tat cag ggg cag 1104 Ala Met Phe Phe Leu Met Lys Gly Thr ProPhe Ile Tyr Gln Gly Gln 355 360 365 gaa ctt ggc atg acc aat acg cat ttcgcc agc ctg gag gat ttt gac 1152 Glu Leu Gly Met Thr Asn Thr His Phe AlaSer Leu Glu Asp Phe Asp 370 375 380 gac gtt gcc gcg aag aaa ctc gcc gttgaa atg cgc cga cag ggc agg 1200 Asp Val Ala Ala Lys Lys Leu Ala Val GluMet Arg Arg Gln Gly Arg 385 390 395 400 gaa gag ccc gag atc ctc gcc ttcctc agc cgc acc ggg cgc gaa aac 1248 Glu Glu Pro Glu Ile Leu Ala Phe LeuSer Arg Thr Gly Arg Glu Asn 405 410 415 tcg cgc acc ccg atg cag tcg gatcag agt gcc cac ggc ggt ttc agc 1296 Ser Arg Thr Pro Met Gln Ser Asp GlnSer Ala His Gly Gly Phe Ser 420 425 430 aat gct acc ccc tgg ttt cct gcgaac agt aat tac cct gta atc aac 1344 Asn Ala Thr Pro Trp Phe Pro Ala AsnSer Asn Tyr Pro Val Ile Asn 435 440 445 gtg gcg gat caa cgt gct gac agcggt tcc gtg ctg aac ttc tat cgt 1392 Val Ala Asp Gln Arg Ala Asp Ser GlySer Val Leu Asn Phe Tyr Arg 450 455 460 gcg ctc att cgc ctg cgc cgg cagatg ccg gta ttg att gaa ggg gct 1440 Ala Leu Ile Arg Leu Arg Arg Gln MetPro Val Leu Ile Glu Gly Ala 465 470 475 480 tat caa ctt ctg ctg ccg acacat ccg cag atc tat gcc tat acc cgt 1488 Tyr Gln Leu Leu Leu Pro Thr HisPro Gln Ile Tyr Ala Tyr Thr Arg 485 490 495 cgt ctg aat gaa cag cag gtgttg gtg atc gtc aat ttc agt gcc cat 1536 Arg Leu Asn Glu Gln Gln Val LeuVal Ile Val Asn Phe Ser Ala His 500 505 510 cag cag gag ata gat ccg caacag ctg tta ctg gac ggc tgg caa ccg 1584 Gln Gln Glu Ile Asp Pro Gln GlnLeu Leu Leu Asp Gly Trp Gln Pro 515 520 525 ctg ctg agc aat tat cag gagcag ggg aaa cgg caa atc tta cgg gct 1632 Leu Leu Ser Asn Tyr Gln Glu GlnGly Lys Arg Gln Ile Leu Arg Ala 530 535 540 tat gag gca caa att tat cagcgg taa 1659 Tyr Glu Ala Gln Ile Tyr Gln Arg 545 550 9 552 PRT Erwiniarhapontici 9 Met Ala Asn Trp Trp Lys Glu Ala Val Ala Tyr Gln Ile Tyr ProArg 1 5 10 15 Ser Phe Asn Asp Ser Asn Asn Asp Gly Ile Gly Asp Leu AsnGly Ile 20 25 30 Thr Glu Lys Leu Asp Tyr Leu Glu Asp Leu Gly Ile Asp LeuIle Trp 35 40 45 Ile Cys Pro Met Tyr Gln Ser Pro Asn Asp Asp Asn Gly TyrAsp Ile 50 55 60 Ser Asp Tyr Gln Lys Ile Met Ala Glu Phe Gly Thr Met AspAsp Phe 65 70 75 80 Asp Arg Leu Leu Glu Gln Val His Ala Arg Gly Met ArgLeu Ile Ile 85 90 95 Asp Leu Val Val Asn His Thr Ser Asp Glu His Pro TrpPhe Leu Ala 100 105 110 Ser Ser Ala Ser Arg Asp Asn Pro Lys Arg Asp TrpTyr Ile Trp Arg 115 120 125 Asp Gly Lys Ala Gly Ala Glu Pro Asn Asn TrpGlu Ser Ile Phe Asn 130 135 140 Gly Ser Ala Trp Lys Tyr Ser Ala Ala ThrGly Gln Tyr Phe Leu His 145 150 155 160 Leu Phe Ser Glu Lys Gln Pro AspLeu Asn Trp Glu Asn Pro Glu Val 165 170 175 Arg Ser Ala Val Tyr Ala MetMet Arg Trp Trp Leu Asp Lys Gly Val 180 185 190 Asp Gly Phe Arg Ile AspAla Ile Cys His Met Lys Lys Glu Pro Thr 195 200 205 Phe Ser Asp Met ProAsn Pro Leu Ala Leu Pro Tyr Val Pro Ser Phe 210 215 220 Glu Arg His LeuAsn Tyr Asp Gly Leu Leu Asp Tyr Val Asp Asp Met 225 230 235 240 Cys GluGln Val Phe Ser His Tyr Asp Ile Val Thr Ile Gly Glu Met 245 250 255 AsnGly Ala Ser Ala Glu Gln Gly Glu Glu Trp Val Gly Glu Gln Arg 260 265 270Gly Arg Leu Asn Met Ile Phe Gln Phe Glu His Val Lys Leu Trp Gln 275 280285 Asp Gly Gln Lys Asp Thr Leu Glu Ala Ser Leu Asp Leu Pro Gly Leu 290295 300 Lys Glu Ile Phe Thr Arg Trp Gln Thr Leu Leu Glu Asn Lys Gly Trp305 310 315 320 Asn Ala Leu Tyr Val Glu Asn His Asp Leu Pro Arg Val ValSer Gly 325 330 335 Trp Gly Asp Asp Lys Asn Tyr Gln Arg Glu Ser Ala ThrAla Ile Ala 340 345 350 Ala Met Phe Phe Leu Met Lys Gly Thr Pro Phe IleTyr Gln Gly Gln 355 360 365 Glu Leu Gly Met Thr Asn Thr His Phe Ala SerLeu Glu Asp Phe Asp 370 375 380 Asp Val Ala Ala Lys Lys Leu Ala Val GluMet Arg Arg Gln Gly Arg 385 390 395 400 Glu Glu Pro Glu Ile Leu Ala PheLeu Ser Arg Thr Gly Arg Glu Asn 405 410 415 Ser Arg Thr Pro Met Gln SerAsp Gln Ser Ala His Gly Gly Phe Ser 420 425 430 Asn Ala Thr Pro Trp PhePro Ala Asn Ser Asn Tyr Pro Val Ile Asn 435 440 445 Val Ala Asp Gln ArgAla Asp Ser Gly Ser Val Leu Asn Phe Tyr Arg 450 455 460 Ala Leu Ile ArgLeu Arg Arg Gln Met Pro Val Leu Ile Glu Gly Ala 465 470 475 480 Tyr GlnLeu Leu Leu Pro Thr His Pro Gln Ile Tyr Ala Tyr Thr Arg 485 490 495 ArgLeu Asn Glu Gln Gln Val Leu Val Ile Val Asn Phe Ser Ala His 500 505 510Gln Gln Glu Ile Asp Pro Gln Gln Leu Leu Leu Asp Gly Trp Gln Pro 515 520525 Leu Leu Ser Asn Tyr Gln Glu Gln Gly Lys Arg Gln Ile Leu Arg Ala 530535 540 Tyr Glu Ala Gln Ile Tyr Gln Arg 545 550 10 27 DNA Erwiniarhapontici 10 ggatccggta ccgttcagca atcaaat 27 11 23 DNA Erwiniarhapontici 11 gtcgacgtct tgccaaaaac ctt 23 12 33 DNA Artificial SequenceA primer 12 ggatccacaa tggcaaccgt tcagcaatca aat 33 13 25 DNA Erwiniarhapontici 13 gtcgacctac gtgattaagt ttata 25 14 27 DNA ArtificialSequence A primer 14 gagatcttgc gcagcacacc gcactgg 27 15 24 DNAArtificial Sequence A primer 15 gtcgactcac agcctctcaa taag 24 16 11 DNAArtificial Sequence A linker sequence 16 accgaattgg g 11 17 27 DNANicotiana tabacum 17 gaattcgttt gacagcttat catcgat 27 18 26 DNANicotiana tabacum 18 ggtaccagct aatttcttta agtaaa 26 19 27 DNAArtificial Sequence A primer 19 gggatccgtg caaactggtg gaaagag 27 20 27DNA Artificial Sequence A primer 20 gtcgacttac cgctgataaa tttgtgc 27 2133 DNA Artificial Sequence A primer 21 ctggtggtca accatgcctc tgacgaacatccc 33 22 33 DNA Artificial Sequence A primer 22 gggatgttcg tcagaggcatggttgaccac cag 33 23 33 DNA Artificial Sequence A primer 23 gagacgtggagcgcagcgcc agaagacgcc ctg 33 24 33 DNA Artificial Sequence A primer 24cagggcgtct tctggcgctg cgctccacgt ctc 33 25 35 DNA Artificial Sequence Aprimer 25 ggtgatcaat aacttcgcgc gagacgctgt gatgc 35 26 35 DNA ArtificialSequence A primer 26 gcatcacagc gtctcgcgcg aagttattga tcacc 35 27 11 PRTErwinia rhapontici 27 Leu Val Val Asn His Thr Ser Asp Glu His Pro 1 5 1028 33 DNA Erwinia rhapontici 28 ctggtggtca accatacctc tgacgaacat ccc 3329 11 PRT Artificial Sequence Site-directed mutagenesis of palatinasefrom Erwinia rhapontici 29 Leu Val Val Asn His Ala Ser Asp Glu His Pro 15 10 30 33 DNA Artificial Sequence Site-directed mutagenesis ofpalatinase from Erwinia rhapontici 30 ctggtggtca accatgcctc tgacgaacatccc 33 31 11 PRT Erwinia rhapontici 31 Glu Thr Trp Ser Ala Thr Pro GluAsp Ala Leu 1 5 10 32 33 DNA Erwinia rhapontic 32 gagacgtgga gcgcaacgccagaagacgcc ctg 33 33 11 PRT Artificial Sequence Site-directedmutagenesis of palatinase from Erwinia rhapontici 33 Glu Thr Trp Ser AlaAla Pro Glu Asp Ala Leu 1 5 10 34 33 DNA Artificial SequenceSite-directed mutagenesis of palatinase from Erwinia rhapontici 34gagacgtgga gcgcagcgcc agaagacgcc ctg 33 35 11 PRT Erwinia rhapontic 35Val Ile Asn Asn Phe Thr Arg Asp Ala Val Met 1 5 10 36 33 DNA Erwiniarhapontic 36 gtgatcaata acttcacgcg agacgctgtg atg 33 37 11 PRTArtificial Sequence Site-directed mutagenesis of palatinase from Erwiniarhapontici 37 Val Ile Asn Asn Phe Ala Arg Asp Ala Val Met 1 5 10 38 33DNA Artificial Sequence Site-directed mutagenesis of palatinase fromErwinia rhapontici 38 gtgatcaata acttcacgcg agacgctgtg atg 33

What is claimed is:
 1. A method for influencing pollen development intransgenic plants by modifying the carbohydrate metabolism, comprisingthe following steps: a) producing a recombinant nucleic acid molecule,comprising the following sequences: regulatory sequences of a promoterthat is active in anthers, in the tapetum and/or in pollen; operativelylinked thereto a DNA sequence which codes for a protein having theenzymatic activity of a sucrose isomerase; and operatively linkedthereto regulatory sequences which can serve as transcription,termination and/or polyadenylation signals in plant cells; b)transferring the nucleic acid molecule from a) to plant cells and c)regenerating transgenic plants.
 2. The recombinant nucleic acidmolecule, comprising a) regulatory sequences of a promoter that isactive in anthers, in the tapetum and/or in pollen; b) operativelylinked thereto a DNA sequence which codes for a protein having theenzymatic activity of a sucrose isomerase; and c) operatively linkedthereto regulatory sequences which can serve as transcription,termination and/or polyadenylation signals in plant cells.
 3. Therecombinant nucleic acid molecule according to claim 2, wherein the DNAsequence originates from Erwinia rhapontici.
 4. The recombinant nucleicacid molecule according to claim 2, wherein the promoter is the T29promoter.
 5. A vector comprising a recombinant nucleic acid moleculeaccording to claim
 2. 6. A microorganism comprising a recombinantnucleic acid molecule according to any one of claims 2 to
 4. 7. Amicroorganism comprising the vector of claim
 5. 8. A method forgenerating male sterile plants comprising the transfer of a recombinantnucleic acid molecule according to any one of claims 2 to 4 to plantcells.
 9. A method for generating male sterile plants comprising thetransfer of the vector of claim 5 to plant cells.
 10. A transgenic plantcell comprising a recombinant nucleic acid molecule according to any oneof claims 2 to
 4. 11. A transgenic plant cell comprising the vector ofclaim
 5. 12. A transgenic plant, protoplast, callus, seed, tuber,cutting, or harvest product comprising the plant cell of claim
 11. 13. Arecombinant nucleic acid molecule comprising a DNA sequence from Erwiniarhapontici or a part thereof that codes for a protein having theenzymatic activity of a palatinase.
 14. A recombinant nucleic acidmolecule according to claim 13, wherein the DNA sequence is SEQ IDNo.
 1. 15. A recombinant nucleic acid molecule containing a DNA sequencefrom Erwinia rhapontici or a part thereof that codes for a proteinhaving the enzymatic activity of a trehalulase.
 16. The recombinantnucleic acid molecule according to claim 15, wherein the DNA sequence isSEQ ID No.
 7. 17. A method for generating male fertile hybrid plants,comprising the following steps: a) producing a first transgenic malesterile parent plant, comprising a nucleic acid molecule which codes fora protein having the enzymatic activity of a sucrose isomerase, b)producing a second transgenic parent plant, comprising a nucleic acidmolecule which codes for a protein having the enzymatic activity of apalatinase and/or a protein having the enzymatic activity of atrehalulase, c) crossing the first parent plant with the second parentplant to generate a hybrid plant, wherein the hybrid plant is malefertile.
 18. A method for generating male fertile hybrid plantscomprising the following steps: a) producing a first transgenic malesterile parent plant comprising a nucleic acid molecule which codes fora protein having the enzymatic activity of a sucrose isomerase, b)producing a second transgenic parent plant comprising a nucleic acidmolecule which codes for a protein which has the biological activity ofa sucrose isomerase inhibitor, c) crossing the first parent plant withthe second parent plant for generating a hybrid plant, wherein thehybrid plant is male fertile.
 19. A method for generating male fertilehybrid plants comprising the following steps: a) producing a firsttransgenic male sterile parent plant comprising a nucleic acid moleculewhich codes for a protein having the enzymatic activity of a sucroseisomerase, b) producing a second transgenic parent plant comprising anucleic acid molecule which codes for a ribozyme which is directedagainst sucrose isomerase mRNA, c) crossing the first parent plant withthe second parent plant for generating a hybrid plant, wherein thehybrid plant is male fertile.
 20. A method for generating male fertilehybrid plants, comprising the following steps: a) producing a firsttransgenic male sterile parent plant comprising a nucleic acid moleculewhich codes for a protein having the enzymatic activity of a sucroseisomerase, b) producing a second transgenic parent plant comprising anucleic acid molecule which codes for a sucrose isomerase antisense orsense RNA, c) crossing the first parent plant with the second parentplant to generate a hybrid plant, wherein the hybrid plant is malefertile.
 21. The method of claim 17, wherein a glycosylation site isinactivated within the protein having the enzymatic activity of apalatinase and/or the protein having the enzymatic activity of atrehalulase due to at least one amino acid exhange in comparison withthe wild type protein.
 22. The recombinant nucleic acid molecule ofclaim 13, wherein a glycosylation site is inactivated within the proteinhaving the enzymatic activity of a palatinase due to at least one aminoacid exchange in comparison with the wild type protein.